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Clonal analyses and gene profiling identify genetic biomarkers of the thermogenic potential of human brown and white preadipocytes.

Xue R, Lynes MD, Dreyfuss JM, Shamsi F, Schulz TJ, Zhang H, Huang TL, Townsend KL, Li Y, Takahashi H, Weiner LS, White AP, Lynes MS, Rubin LL, Goodyear LJ, Cypess AM, Tseng YH - Nat. Med. (2015)

Bottom Line: Knocking out the positive UCP1 regulators, PREX1 and EDNRB, in brown preadipocytes using CRISPR-Cas9 markedly abolished the high level of UCP1 in brown adipocytes differentiated from the preadipocytes.Finally, we were able to prospectively isolate adipose progenitors with great thermogenic potential using the cell surface marker CD29.These data provide new insights into the cellular heterogeneity in human fat and offer potential biomarkers for identifying thermogenically competent preadipocytes.

View Article: PubMed Central - PubMed

Affiliation: 1] Section on Integrative Physiology and Metabolism, Research Division, Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts, USA. [2] Division of Endocrinology and Metabolism, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China.

ABSTRACT
Targeting brown adipose tissue (BAT) content or activity has therapeutic potential for treating obesity and the metabolic syndrome by increasing energy expenditure. However, both inter- and intra-individual differences contribute to heterogeneity in human BAT and potentially to differential thermogenic capacity in human populations. Here we generated clones of brown and white preadipocytes from human neck fat and characterized their adipogenic and thermogenic differentiation. We combined an uncoupling protein 1 (UCP1) reporter system and expression profiling to define novel sets of gene signatures in human preadipocytes that could predict the thermogenic potential of the cells once they were maturated. Knocking out the positive UCP1 regulators, PREX1 and EDNRB, in brown preadipocytes using CRISPR-Cas9 markedly abolished the high level of UCP1 in brown adipocytes differentiated from the preadipocytes. Finally, we were able to prospectively isolate adipose progenitors with great thermogenic potential using the cell surface marker CD29. These data provide new insights into the cellular heterogeneity in human fat and offer potential biomarkers for identifying thermogenically competent preadipocytes.

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Clonal analysis of human brown and white fat progenitors. The strategy of clonal analysis of hWAT-SVF and hBAT-SVF progenitors is shown as a dendrogram. 152 clones from hWAT-SVF and 128 clones from hBAT-SVF were derived by limiting dilution from 4 subjects. Adipogenic capacity was determined by Nile red staining and UCP1 level was determined by luciferase activity on day 18. Detailed selection criteria are described in Supplementary Figures 6 and 7. Selected highly adipogenic clones (adipogenic ++) were pre-treated with 3.3 nM BMP7 for 6 days and then differentiated into mature adipocytes in a 96-well plate. Luciferase activity was measured on day 18 and divided into different levels (negative, Neg; low; medium, Med; high) after normalized to protein content. The positive response (+) to BMP7 pretreatment was defined by more than 1.5-fold increase of luciferase activity between BMP7-pretreated and vehicle groups.
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Figure 3: Clonal analysis of human brown and white fat progenitors. The strategy of clonal analysis of hWAT-SVF and hBAT-SVF progenitors is shown as a dendrogram. 152 clones from hWAT-SVF and 128 clones from hBAT-SVF were derived by limiting dilution from 4 subjects. Adipogenic capacity was determined by Nile red staining and UCP1 level was determined by luciferase activity on day 18. Detailed selection criteria are described in Supplementary Figures 6 and 7. Selected highly adipogenic clones (adipogenic ++) were pre-treated with 3.3 nM BMP7 for 6 days and then differentiated into mature adipocytes in a 96-well plate. Luciferase activity was measured on day 18 and divided into different levels (negative, Neg; low; medium, Med; high) after normalized to protein content. The positive response (+) to BMP7 pretreatment was defined by more than 1.5-fold increase of luciferase activity between BMP7-pretreated and vehicle groups.

Mentions: In order to study homogenous cell populations derived from human adipose precursor cells, we isolated a total of 280 clonal preadipocyte cell lines (152 hWAT-SVF clones and 128 hBAT-SVF clones) from the immortalized pool populations of all four subjects. Of these lines, 44% (67 out of 152) of hWAT-SVF and 70% (90 out of 128) of hBAT-SVF clones robustly differentiated into mature adipocytes (Fig. 3 and Supplementary Fig. 6). To determine UCP1 induction in the differentiated state, we measured luciferase reporter activity in each clonal line after 18-days of adipogenic differentiation (Supplementary Fig. 7). Importantly, the data revealed that up to 96% of the hWAT-SVF clones were UCP1 negative, while more than 94% of the hBAT-SVF clones displayed differential levels of UCP1-luciferase activities (Fig. 3).


Clonal analyses and gene profiling identify genetic biomarkers of the thermogenic potential of human brown and white preadipocytes.

Xue R, Lynes MD, Dreyfuss JM, Shamsi F, Schulz TJ, Zhang H, Huang TL, Townsend KL, Li Y, Takahashi H, Weiner LS, White AP, Lynes MS, Rubin LL, Goodyear LJ, Cypess AM, Tseng YH - Nat. Med. (2015)

Clonal analysis of human brown and white fat progenitors. The strategy of clonal analysis of hWAT-SVF and hBAT-SVF progenitors is shown as a dendrogram. 152 clones from hWAT-SVF and 128 clones from hBAT-SVF were derived by limiting dilution from 4 subjects. Adipogenic capacity was determined by Nile red staining and UCP1 level was determined by luciferase activity on day 18. Detailed selection criteria are described in Supplementary Figures 6 and 7. Selected highly adipogenic clones (adipogenic ++) were pre-treated with 3.3 nM BMP7 for 6 days and then differentiated into mature adipocytes in a 96-well plate. Luciferase activity was measured on day 18 and divided into different levels (negative, Neg; low; medium, Med; high) after normalized to protein content. The positive response (+) to BMP7 pretreatment was defined by more than 1.5-fold increase of luciferase activity between BMP7-pretreated and vehicle groups.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496292&req=5

Figure 3: Clonal analysis of human brown and white fat progenitors. The strategy of clonal analysis of hWAT-SVF and hBAT-SVF progenitors is shown as a dendrogram. 152 clones from hWAT-SVF and 128 clones from hBAT-SVF were derived by limiting dilution from 4 subjects. Adipogenic capacity was determined by Nile red staining and UCP1 level was determined by luciferase activity on day 18. Detailed selection criteria are described in Supplementary Figures 6 and 7. Selected highly adipogenic clones (adipogenic ++) were pre-treated with 3.3 nM BMP7 for 6 days and then differentiated into mature adipocytes in a 96-well plate. Luciferase activity was measured on day 18 and divided into different levels (negative, Neg; low; medium, Med; high) after normalized to protein content. The positive response (+) to BMP7 pretreatment was defined by more than 1.5-fold increase of luciferase activity between BMP7-pretreated and vehicle groups.
Mentions: In order to study homogenous cell populations derived from human adipose precursor cells, we isolated a total of 280 clonal preadipocyte cell lines (152 hWAT-SVF clones and 128 hBAT-SVF clones) from the immortalized pool populations of all four subjects. Of these lines, 44% (67 out of 152) of hWAT-SVF and 70% (90 out of 128) of hBAT-SVF clones robustly differentiated into mature adipocytes (Fig. 3 and Supplementary Fig. 6). To determine UCP1 induction in the differentiated state, we measured luciferase reporter activity in each clonal line after 18-days of adipogenic differentiation (Supplementary Fig. 7). Importantly, the data revealed that up to 96% of the hWAT-SVF clones were UCP1 negative, while more than 94% of the hBAT-SVF clones displayed differential levels of UCP1-luciferase activities (Fig. 3).

Bottom Line: Knocking out the positive UCP1 regulators, PREX1 and EDNRB, in brown preadipocytes using CRISPR-Cas9 markedly abolished the high level of UCP1 in brown adipocytes differentiated from the preadipocytes.Finally, we were able to prospectively isolate adipose progenitors with great thermogenic potential using the cell surface marker CD29.These data provide new insights into the cellular heterogeneity in human fat and offer potential biomarkers for identifying thermogenically competent preadipocytes.

View Article: PubMed Central - PubMed

Affiliation: 1] Section on Integrative Physiology and Metabolism, Research Division, Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts, USA. [2] Division of Endocrinology and Metabolism, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China.

ABSTRACT
Targeting brown adipose tissue (BAT) content or activity has therapeutic potential for treating obesity and the metabolic syndrome by increasing energy expenditure. However, both inter- and intra-individual differences contribute to heterogeneity in human BAT and potentially to differential thermogenic capacity in human populations. Here we generated clones of brown and white preadipocytes from human neck fat and characterized their adipogenic and thermogenic differentiation. We combined an uncoupling protein 1 (UCP1) reporter system and expression profiling to define novel sets of gene signatures in human preadipocytes that could predict the thermogenic potential of the cells once they were maturated. Knocking out the positive UCP1 regulators, PREX1 and EDNRB, in brown preadipocytes using CRISPR-Cas9 markedly abolished the high level of UCP1 in brown adipocytes differentiated from the preadipocytes. Finally, we were able to prospectively isolate adipose progenitors with great thermogenic potential using the cell surface marker CD29. These data provide new insights into the cellular heterogeneity in human fat and offer potential biomarkers for identifying thermogenically competent preadipocytes.

Show MeSH
Related in: MedlinePlus