Human fetal mesenchymal stem cells differentiate into brown and white adipocytes: a role for ERRalpha in human UCP1 expression

Abstract

We investigated the ability of fetal mesenchymal stem cells (fMSCs) to differentiate into brown and white adipocytes and compared the expression of a number of marker genes and key regulatory factors. We showed that the expression of key adipocyte regulators and markers during differentiation is similar to that in other human and murine adipocyte models, including induction of PPARgamma2 and FABP4. Notably, we found that the preadipocyte marker, Pref-1, is induced early in differentiation and then declines markedly as the process continues, suggesting that fMSCs first acquire preadipocyte characteristics as they commit to the adipogenic lineage, prior to their differentiation into mature adipocytes. After adipogenic induction, some stem cell isolates differentiated into cells resembling brown adipocytes and others into white adipocytes. Detailed investigation of one isolate showed that the novel brown fat-determining factor PRDM16 is expressed both before and after differentiation. Importantly, these cells exhibited elevated basal UCP-1 expression, which was dependent on the activity of the orphan nuclear receptor ERRalpha, highlighting a novel role for ERRalpha in human brown fat. Thus fMSCs represent a useful in vitro model for human adipogenesis, and provide opportunities to study the stages prior to commitment to the adipocyte lineage. They also offer invaluable insights into the characteristics of human brown fat.

Fig 1

Fetal Mesenchymal stem cells differentiate into adipocytes A) Fetal Mesenchymal Stem Cells before (1) and after differentiation. into adipocytes and bone as demonstrated by light microscopy (2), Oil Red O staining to demonstrate lipid accumulation (3) or Von Kossa staining to demonstrate mineralisation (4) (400x magnification). B) Time course of mRNA expression of key markers and regulators of adipocytes by quantitative PCR. C= Confluent cells, +6 = 6 hours post hormonal induction of differentiation, all other time points indicate days post induction. Fetal Blood and Fetal Bone Marrow indicate the source of isolate. Error bars indicate SD. Relative expression in arbitrary units.

Fig 2

Two different isolates of fetal mesenchymal stem cells differentiate into adipocytes which resemble white and brown adipocytes respectively. A) UCP-1 mRNA expression in fMSC 10+1 and 9+4 isolates after 21 days of differentiation. B) UCP-1 mRNA expression in fMSC 9+4 isolatre before and after 21 days of differentiation, and differentiated human adult sub-cutaneous adipocytes (hPA). nd = not detected. C and D) mRNA expression of PGC1α and RIP140 (C) and PRDM16 (D) in the two isolates before and after differentiation. ** indicates p<0.001, * p<0.05 for comparison between the two isolates. E) Expression of UCP-1 mRNA in 9+4 isolate after 21 days differentiation, before and after treatment with 10μM Isoproteranol for 5 hours. Error bars indicate SD. nd indicates not detected. Relative expression in arbitrary units.

Fig 3

Immunophenotyping confirms Mesenchymal Stem Cell Charecterisitics Undifferentiated fMSC 9+4 and 10+1 cells express SH2, SH3 and Vimentin, but not CD14, CD 34 or CD45, confirming there mesenchyme origin.

Fig 4

UCP-1 expression in fMSC 9+4 adipocytes can be induced by Rosiglitazone treatment, and reduced by the ERRα inverse agonist. mRNA expression determined by qPCR A)UCP-1 expression following Rosiglitazone or XCT790 treatment for 48 hours. * p=<0.05, ** = p<0.005. B)UCP-1 expression after 48 hour treatment with PPARg anatagonist G3335 * = p<0.05. C) Chromatin Immunoprecipitation showing ERRa bound to the UCP1 enhancer element in differentiated 9+4 fMSCs, but not to a region in the coding sequence of the same gene. D) Reporter assay using this construct in Cos-1 cells. ERRa activates the enhancer, and its activity is further trans-activated by PGC1a and repressed by RIP140. The ERRa inverse agonist XCT790 prevents this induction. E) ERRα expression in 10+1 and 9+4 isolates before and after 21 days adipogenic differentiation. mRNA expression by qPCR. Error Bars indicate SD.