ABSTRACT
In this chapter, we describe a protocol to induce at a high rate the differentiation of brown/brown like adipocyte progenitors (BAPs) derived from human induced pluripotent stem cells (hiPSCs). We also describe culture conditions to maintain hiPSCs and to derive hiPSC-BAPs.This novel culture system provides an unlimited source of human brown adipocytes and a unique means for studying events regulating the generation and recruitment of human BAPs.
Subject(s)
Adipocytes, Brown/cytology , Cell Differentiation/drug effects , Cellular Reprogramming/drug effects , Induced Pluripotent Stem Cells/cytology , Amides/pharmacology , Animals , Cell Line , Cell Survival/drug effects , Feeder Cells/drug effects , Fibroblasts/drug effects , Humans , Mice , Mitomycin/pharmacology , Pyridines/pharmacologyABSTRACT
Human induced pluripotent stem cells (hiPSCs) show great promise for obesity treatment as they represent an unlimited source of brown/brite adipose progenitors (BAPs). However, hiPSC-BAPs display a low adipogenic capacity compared to adult-BAPs when maintained in a traditional adipogenic cocktail. The reasons of this feature are unknown and hamper their use both in cell-based therapy and basic research. Here we show that treatment with TGFß pathway inhibitor SB431542 together with ascorbic acid and EGF were required to promote hiPSCs-BAP differentiation at a level similar to adult-BAP differentiation. hiPSC-BAPs expressed the molecular identity of adult-UCP1 expressing cells (PAX3, CIDEA, DIO2) with both brown (ZIC1) and brite (CD137) adipocyte markers. Altogether, these data highlighted the critical role of TGFß pathway in switching off hiPSC-brown adipogenesis and revealed novel factors to unlock their differentiation. As hiPSC-BAPs display similarities with adult-BAPs, it opens new opportunities to develop alternative strategies to counteract obesity.
Subject(s)
Adipocytes, Brown/metabolism , Induced Pluripotent Stem Cells/metabolism , Signal Transduction/genetics , Transforming Growth Factor beta/antagonists & inhibitors , Adipocytes, Brown/cytology , Adipocytes, Brown/drug effects , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Ascorbic Acid/pharmacology , Benzamides/pharmacology , Biomarkers/metabolism , Cell Differentiation/drug effects , Cell Line , Dioxoles/pharmacology , Epidermal Growth Factor/pharmacology , Gene Expression , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/drug effects , Iodide Peroxidase/genetics , Iodide Peroxidase/metabolism , PAX3 Transcription Factor/genetics , PAX3 Transcription Factor/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolism , Tumor Necrosis Factor Receptor Superfamily, Member 9/genetics , Tumor Necrosis Factor Receptor Superfamily, Member 9/metabolism , Iodothyronine Deiodinase Type IIABSTRACT
Mesenchymal stem cells (MSCs) derived from human induced pluripotent stem cells (hiPSCs) provide a novel source for generating adipocytes, thus opening new avenues for fundamental research and clinical medicine. We present the adipogenic potential of hiPSCs and the various methods to derive hiPSC-MSCs. We discuss the main characteristic of hiPSC-MSCs, which is their low adipogenic capacity as compared to adult-MSCs. Finally, we propose several hypotheses to explanation this feature, underlying a potential critical role of the micro-environment. We favour the hypothesis that the range of factors or culture conditions required to induce adipocyte differentiation of MSCs derived from adult tissues and from embryonic-like cells could differ.
ABSTRACT
OBJECTIVE: To characterize brown adipose tissue (BAT) in the human perirenal adipose tissue depot. METHOD: Perirenal adipose tissue biopsies were obtained from 55 healthy kidney donors. Expression analysis was performed using microarray, real-time PCR, immunoblotting and immunohistochemistry. Additional studies using human stem cells were performed. RESULTS: UCP1 gene expression analysis revealed a large intra-individual variation in the perirenal adipose tissue biopsies. Both multi- and unilocular UCP1-positive adipocytes were detected in several of the adipose tissue samples analyzed by immunohistochemical staining. Microarray analysis identified 54 genes that were overexpressed in UCP1-positive perirenal adipose tissue. Real-time PCR analysis of BAT candidate genes revealed a set of genes that were highly correlated to UCP1 and a set of three transcription factor genes (PRDM16, PGC1α, and RXRγ) that were highly correlated to each other. RXRγ displayed nuclear immunoreactivity in brown adipocytes and an increased gene expression during brown adipogenesis in human stem cells. CONCLUSION: Our data provides the first molecular characterization of BAT in the perirenal adipose tissue depot. Furthermore, it highlights the transcription factor RXRγ as a new player in BAT development.
Subject(s)
Adipocytes, Brown/metabolism , Adipose Tissue, Brown/metabolism , Ion Channels/metabolism , Mitochondrial Proteins/metabolism , Retinoid X Receptor gamma/metabolism , Adipogenesis , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Humans , Immunohistochemistry , Ion Channels/genetics , Kidney , Mitochondrial Proteins/genetics , Oligonucleotide Array Sequence Analysis , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Retinoid X Receptor gamma/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , Uncoupling Protein 1ABSTRACT
Identification of molecular mechanisms involved in generation of different types of adipocytes is progressing substantially in mice. However, much less is known regarding characterization of brown (BAP) and white adipocyte progenitors (WAPs) in humans, highlighting the need for an in vitro model of human adipocyte development. Here, we report a procedure to selectively derive BAP and WAPs from human-induced pluripotent stem cells. Molecular characterization of APs of both phenotypes revealed that BMP4, Hox8, Hoxc9, and HoxA5 genes were specifically expressed in WAPs, whereas expression of PRDM16, Dio2, and Pax3 marked BAPs. We focused on Pax3 and we showed that expression of this transcription factor was enriched in human perirenal white adipose tissue samples expressing UCP1 and in human classical brown fat. Finally, functional experiments indicated that Pax3 was a critical player of human AP fate as its ectopic expression led to convert WAPs into brown-like APs. Together, these data support a model in which Pax3 is a new marker of human BAPs and a molecular mediator of their fate. The findings of this study could lead to new anti-obesity therapies based on the recruitment of APs and constitute a platform for investigating in vitro the developmental origins of human white and brown adipocytes.
Subject(s)
Adipocytes, Brown/cytology , Adipocytes, White/cytology , Cell Differentiation , Induced Pluripotent Stem Cells/cytology , Paired Box Transcription Factors/metabolism , Adipocytes, Brown/drug effects , Adipocytes, Brown/metabolism , Adipocytes, White/drug effects , Adipocytes, White/metabolism , Adipogenesis/drug effects , Aged, 80 and over , Animals , Cell Differentiation/drug effects , Female , Humans , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/metabolism , Male , Mice , PAX3 Transcription Factor , Phenotype , Tretinoin/pharmacologyABSTRACT
Human adipose-derived stem cell populations express cell surface markers such as CD105, CD73, CD146 and CD140a/PDFGRα. However, it was unclear whether these markers could discriminate subpopulations of undifferentiated cells and whether the expression of these markers is modulated during differentiation. To address this issue, we analysed the immunophenotype of cultured human multipotent adipose derived stem (hMADS) cell populations at different adipocyte differentiation steps. We found that 100% of undifferentiated cells expressed CD73 and CD105. In contrast, CD146 and CD140a/PDFGRα marked two different subpopulations of cells. CD140a/PDGFRα subpopulation was regulated by FGF2, a critical factor of human adipose-derived stem cell self-renewal. During differentiation, CD73 was maintained and marked lipid-laden cells, whereas CD105 expression was inhibited in fully differentiated cells. The percentage of CD146 and CD140a/PDFGRα-positive cells declined as soon as cells had undergone differentiation. Altogether, these data support the notion that expanded adipose-derived stem cells are heterogeneous mixtures of cells and cell surface markers studied can discriminate subpopulations.