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Development of an OP9 derived cell line as a robust model to rapidly study adipocyte differentiation.

Lane JM, Doyle JR, Fortin JP, Kopin AS, Ordovás JM - PLoS ONE (2014)

Bottom Line: We implemented a method to quantify lipid accumulation using automated microscopy and tested the ability of our model to detect alterations in lipid accumulation by reducing levels of the known master adipogenic regulator Pparγ.We determine that reduction in levels of Spi1 leads to an increase in lipid accumulation.Together with our microscopy based lipid accumulation assay, adipogenesis assays can be achieved in just four days' time.

View Article: PubMed Central - PubMed

Affiliation: Massachusetts General Hospital, Center for Human Genetics Research, 185 Cambridge Street, Boston, MA 02114, United States of America; Division of Sleep Medicine, Brigham and Women's Hospital, 75 Francis St, Boston, MA 02115, United States of America; Jean Mayer-USDA Human Nutrition Research Center on Aging at Tufts University, Boston, MA 02111, United States of America.

ABSTRACT
One hallmark of obesity is adipocyte hypertrophy and hyperplasia. To gain novel insights into adipose biology and therapeutics, there is a pressing need for a robust, rapid, and informative cell model of adipocyte differentiation for potential RNAi and drug screens. Current models are prohibitive for drug and RNAi screens due to a slow differentiation time course and resistance to transfection. We asked if we could create a rapid, robust model of adipogenesis to potentially enable rapid functional and obesity therapeutic screens. We generated the clonal population OP9-K, which differentiates rapidly and reproducibly, and displays classic adipocyte morphology: rounded cell shape, lipid accumulation, and coalescence of lipids into a large droplet. We further validate the OP9-K cells as an adipocyte model system by microarray analysis of the differentiating transcriptome. OP9-K differentiates via known adipogenic pathways, involving the transcriptional activation and repression of common adipose markers Plin1, Gata2, C/Ebpα and C/Ebpβ and biological pathways, such as lipid metabolism, PPARγ signaling, and osteogenesis. We implemented a method to quantify lipid accumulation using automated microscopy and tested the ability of our model to detect alterations in lipid accumulation by reducing levels of the known master adipogenic regulator Pparγ. We further utilized our model to query the effects of a novel obesity therapeutic target, the transcription factor SPI1. We determine that reduction in levels of Spi1 leads to an increase in lipid accumulation. We demonstrate rapid, robust differentiation and efficient transfectability of the OP9-K cell model of adipogenesis. Together with our microscopy based lipid accumulation assay, adipogenesis assays can be achieved in just four days' time. The results of this study can contribute to the development of rapid screens with the potential to deepen our understanding of adipose biology and efficiently test obesity therapeutics.

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The transcriptome of OP9 clone K cells is analogous to previous models of adipogenesis.Mid and late stage known adipocyte markers are confirmed during OP9 cell adipogenesis. Quantitative PCR of adipocyte marker genes at 0, 24, 48, and 72 hrs after addition of IO media. Standard deviation is derived from 3 replicates.
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pone-0112123-g002: The transcriptome of OP9 clone K cells is analogous to previous models of adipogenesis.Mid and late stage known adipocyte markers are confirmed during OP9 cell adipogenesis. Quantitative PCR of adipocyte marker genes at 0, 24, 48, and 72 hrs after addition of IO media. Standard deviation is derived from 3 replicates.

Mentions: To evaluate if OP9 cells undergo a differentiation process similar to other adipocyte models, a microarray was performed on RNA extracted from OP9 clone K cells during the course of differentiation (Appendices S2-S13 in Files S1 and S2). RNA from clone K cells treated for 0, 24, 48, or 72 hours with IO media was collected (corresponding to day 0, 1, 2, and 3). Samples were hybridized to the Affymetrix GeneChip Mouse Gene 1.0 ST Array and analyzed using Expression Console from Affymetrix and the R Limma package. To verify the results of the microarray, quantitative PCR (qPCR) was performed on four known adipose marker genes: Plin1, Gata2, C/Ebpα and C/Ebpβ (Figure 2). Microarray and qPCR demonstrate C/Ebpα and Plin1 are upregulated and Gata2 is downregulated during OP9 adipogenesis (Figure 2 and Table 1). C/Ebpβ is expressed in OP9 cells during adipogenesis, but levels do not significantly change during the course of differentiation. This corroborates protein expression data from Wolins et al. suggesting that OP9 cells are later stage preadipocytes [25]. Tables S1 and S2 show the 25 most up/down regulated genes from each time point.


Development of an OP9 derived cell line as a robust model to rapidly study adipocyte differentiation.

Lane JM, Doyle JR, Fortin JP, Kopin AS, Ordovás JM - PLoS ONE (2014)

The transcriptome of OP9 clone K cells is analogous to previous models of adipogenesis.Mid and late stage known adipocyte markers are confirmed during OP9 cell adipogenesis. Quantitative PCR of adipocyte marker genes at 0, 24, 48, and 72 hrs after addition of IO media. Standard deviation is derived from 3 replicates.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4237323&req=5

pone-0112123-g002: The transcriptome of OP9 clone K cells is analogous to previous models of adipogenesis.Mid and late stage known adipocyte markers are confirmed during OP9 cell adipogenesis. Quantitative PCR of adipocyte marker genes at 0, 24, 48, and 72 hrs after addition of IO media. Standard deviation is derived from 3 replicates.
Mentions: To evaluate if OP9 cells undergo a differentiation process similar to other adipocyte models, a microarray was performed on RNA extracted from OP9 clone K cells during the course of differentiation (Appendices S2-S13 in Files S1 and S2). RNA from clone K cells treated for 0, 24, 48, or 72 hours with IO media was collected (corresponding to day 0, 1, 2, and 3). Samples were hybridized to the Affymetrix GeneChip Mouse Gene 1.0 ST Array and analyzed using Expression Console from Affymetrix and the R Limma package. To verify the results of the microarray, quantitative PCR (qPCR) was performed on four known adipose marker genes: Plin1, Gata2, C/Ebpα and C/Ebpβ (Figure 2). Microarray and qPCR demonstrate C/Ebpα and Plin1 are upregulated and Gata2 is downregulated during OP9 adipogenesis (Figure 2 and Table 1). C/Ebpβ is expressed in OP9 cells during adipogenesis, but levels do not significantly change during the course of differentiation. This corroborates protein expression data from Wolins et al. suggesting that OP9 cells are later stage preadipocytes [25]. Tables S1 and S2 show the 25 most up/down regulated genes from each time point.

Bottom Line: We implemented a method to quantify lipid accumulation using automated microscopy and tested the ability of our model to detect alterations in lipid accumulation by reducing levels of the known master adipogenic regulator Pparγ.We determine that reduction in levels of Spi1 leads to an increase in lipid accumulation.Together with our microscopy based lipid accumulation assay, adipogenesis assays can be achieved in just four days' time.

View Article: PubMed Central - PubMed

Affiliation: Massachusetts General Hospital, Center for Human Genetics Research, 185 Cambridge Street, Boston, MA 02114, United States of America; Division of Sleep Medicine, Brigham and Women's Hospital, 75 Francis St, Boston, MA 02115, United States of America; Jean Mayer-USDA Human Nutrition Research Center on Aging at Tufts University, Boston, MA 02111, United States of America.

ABSTRACT
One hallmark of obesity is adipocyte hypertrophy and hyperplasia. To gain novel insights into adipose biology and therapeutics, there is a pressing need for a robust, rapid, and informative cell model of adipocyte differentiation for potential RNAi and drug screens. Current models are prohibitive for drug and RNAi screens due to a slow differentiation time course and resistance to transfection. We asked if we could create a rapid, robust model of adipogenesis to potentially enable rapid functional and obesity therapeutic screens. We generated the clonal population OP9-K, which differentiates rapidly and reproducibly, and displays classic adipocyte morphology: rounded cell shape, lipid accumulation, and coalescence of lipids into a large droplet. We further validate the OP9-K cells as an adipocyte model system by microarray analysis of the differentiating transcriptome. OP9-K differentiates via known adipogenic pathways, involving the transcriptional activation and repression of common adipose markers Plin1, Gata2, C/Ebpα and C/Ebpβ and biological pathways, such as lipid metabolism, PPARγ signaling, and osteogenesis. We implemented a method to quantify lipid accumulation using automated microscopy and tested the ability of our model to detect alterations in lipid accumulation by reducing levels of the known master adipogenic regulator Pparγ. We further utilized our model to query the effects of a novel obesity therapeutic target, the transcription factor SPI1. We determine that reduction in levels of Spi1 leads to an increase in lipid accumulation. We demonstrate rapid, robust differentiation and efficient transfectability of the OP9-K cell model of adipogenesis. Together with our microscopy based lipid accumulation assay, adipogenesis assays can be achieved in just four days' time. The results of this study can contribute to the development of rapid screens with the potential to deepen our understanding of adipose biology and efficiently test obesity therapeutics.

Show MeSH
Related in: MedlinePlus