Thermogenic adipocyte generation using human pluripotent stem cells

Introduction

The development of obesity and associated metabolic disorders such as diabetes and heart diseases is a major health issue. Obesity results from an imbalance between calorie intake and energy expenditure. The scientific community is focusing attention on white adipose tissue (WAT) that stores energy, and on means to fight its expansion. However, modern lifestyles are often not compatible with a reduction in energy intake. Current anti-obesity drugs to reduce energy intake may have major side effects for the patients. Bariatric surgery has proven efficient for obesity, although long-term complications and obesity relapse may occur. The identification of new anti-obesity targets is thus urgently required. In contrast to WAT, classical brown adipocytes and brown-like adipocytes (BAs) dispersed in WATs, mainly in subcutaneous fat depots, are specialized in energy expenditure thanks to their high content of mitochondria expressing the uncoupling protein-1 (UCP1). Upon activation, BAs consume metabolic substrates and burn fat and sugars via uncoupling of oxidative phosphorylation, in turn inhibiting ATP synthesis. The ability of BAs to actively drain circulating glucose and triglycerides to oxidize them can prevent hyperglycemia and hypertriglyceridemia. BAs secrete adipokines that may also contribute to metabolic effects. BAs are therefore promising cell targets to counteract obesity and type-2 diabetes. However, major obstacles hamper BA-based treatment of obesity, including the scarcity of BAs in adult humans.

How to Increase the Mass of Brown-Like Adipocytes in Obese Patients?

Brown adipocytes present at birth persist only around deep organs in healthy adult humans. In addition, brown adipose tissue (BA) activity is lower in overweight and obese individuals than in lean ones. The proof-of-concept of the beneficial effects of brown fat transplantation has been achieved in rodents, where normoglycemia was restored in diabetic mice and obesity reduced in Ob/Ob mice.

This has given rise to the notion of increasing the brown adipose tissue (BA) mass in obese patients as a therapeutic approach to counteract obesity and its associated metabolic complications. A challenge now is to identify an abundant source of human brown adipocyte progenitors (BAPs) for transplantation.

The generation of induced pluripotent stem cells from obese patients as an unlimited source of BAPs that could be expanded for autologous transplantation is a recently discussed option [and see below]. Another option that we discuss in the present review is to promote endogenous BA generation in obese patients. Understanding the mechanisms governing the commitment of human pluripotent stem cells toward the brown-like adipogenic lineage, as well as the differentiation of BAPs into functional BAs, should help addressing this issue.

Human Cell Models Available for Investigating Brown-Like Adipocyte Biology

The identification of molecular mechanisms involved in thermogenic adipocyte generation is progressing substantially in mice. However, much less is known in humans, thus highlighting the need for an in vitro model of human adipocyte development. Because of the rareness of BAs in adult humans, immortalized cell lines or multipotent stem cells derived from adipose tissues of young donors (hMADS cells) are the main cellular models used to identify pathways critical for adipogenesis. hMADS cell lines have been isolated from adipose tissues of young donors in our laboratory. They are not immortalized cells, but can be maintained for several passages in vitro thanks to the intrinsic high self-renewal capacity of stem cells. Interestingly, hMADS cells can be converted into functional brown-like adipocytes. However, the features of infant hMADS dramatically decrease with aging. In addition, these cells are already committed in the adipose lineage, thus precluding the possibility of investigating the earliest steps of adipogenesis.

Pluripotent Stem Cells Represent a Powerful Model to Identify Pathways Governing Thermogenic Adipocyte Development

Pluripotent stem cells (PSCs), i.e., embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), display a quasi-unlimited self-renewal capacity and are an abundant source of multiple cell types of therapeutic interest. Some papers in the early 2000s reported the potential of human ES cells to generate adipocytes. These observations suggested that PSCs could be a valuable tool to identify pathways regulating the different steps of adipogenesis, i.e., from the generation of adipose progenitors to their differentiation into mature adipocytes. Then, Taura et al. demonstrated that human iPSCs have an adipogenic potential comparable to that of human ES cells. However, these authors did not address the adipogenesis efficiency and the phenotype of adipocytes generated. Surprisingly, a cocktail of hematopoietic factors allowed Nishio and colleagues to report, for the first time, the capacity of human iPSCs to generate substantial brown adipocytes [BAs]. These findings support the idea that, as previously shown in mice, the BMP signaling pathway plays a critical role in human brown adipocyte generation. However, Nishio did not purify BAPs from differentiating hiPSCs and there was no evidence that the stem cells progressed through a complete adipogenic program to generate adipocytes. Ahfeldt et al. purified hiPSC-derived fibroblasts that were able to undergo differentiation into white adipocytes or BAs following forced expression of adipogenic master genes. This strategy allows the generation of human BAs and may be a powerful tool for drug discovery, but the question arises as to whether these cells with ectopic expression of adipogenic master genes faithfully reflect physiological adipogenesis. More recently, a procedure to isolate expandable BAPs from hiPSCs and to generate high levels of functional BAs with no gene transfer was described. West and colleagues clonally derived several white- and brown- adipocyte progenitors from hES cell lines and assessed their adipogenic potential when encapsulated in a biocompatible matrix approved for use in human clinical studies. These models provide an opportunity to make effective use of hiPSC features to identify critical pathways governing the development of brown-like adipocytes.

Human Pluripotent Stem Cell Commitment Toward the Brown-Like Adipogenic Lineage is Negatively Regulated by the Retinoic Acid Pathway

Mohsen-Kanson and colleagues, in our laboratory, investigated factors involved in the commitment of pluripotent stem cells toward adipogenic lineages. Four hiPS cell lines and one hES cell line were studied for that purpose. Adipogenic markers, including UCP1, Dio2, PGC1α, and PRDM16, were detected in differentiated cultures, indicating that cells having a brown-like adipocyte gene program were spontaneously generated during differentiation. However, the adipogenesis efficiency was weak. Indeed, adipocytes were co-stained with LipidTox (for triglyceride staining) and CD73 (an adipocyte cell surface marker), and then quantified by flow cytometry. The data showed that the number of LipidTox⁺/CD73⁺ cells represented only 2% of cells in the differentiated cultures. Small-scale drug screening to uncover signaling pathways regulating the earliest steps of human adipogenesis revealed that the retinoic acid (RA) pathway promoted hiPSCS commitment toward the adipogenic lineage by increasing the number of LipidTox⁺/CD73⁺ cells to 15%. In contrast, expression of the brown adipocyte specific marker UCP1 was inhibited in RA-treated cultures. Together, these data support the hypothesis that RA pathway activation at an early development stage dramatically promotes the differentiation of human PSCs into the UCP1-negative adipocyte lineage, while inhibiting UCP1-positive adipocyte generation (see Figure 1). This observation is reminiscent of the critical role of RA in the early steps of mouse ES cell white adipogenesis. The identification of RA targets could provide a means to uncover genes involved in the earliest steps of adipogenesis. The combination of computational and experimental approaches in mouse ES cells revealed an extensive network of transcription factors that might coordinate the expression of genes essential for the acquisition of adipocyte characteristics. This could represent a unique comprehensive resource that could be further explored to investigate human adipocyte development.

Source: frontiersin.org/articles/10.3389/fendo.2019.00932/full Note: Content may be edited for style and length.