Analyses and Utilization of Selectively Tuned Human Adipose-Derived Stromal/Stem Cell Exosomes.

We have developed a hollow fiber bioreactor-based production system for manufacturing large quantities of extracellular vesicles (EVs) containing exosomes from adult human adipose-derived stromal/stem cells (ASCs). By manipulating the cellular bioreactor environment, we have found that we can alter ASC EV production, secretion, and surface protein composition. The aims of this chapter are to describe the methodology for culturing and tuning of adipose ASCs in a bioreactor, along with the collection and isolation of the EVs containing exosomes demonstrating increased HSP70 content.

Biguanides Induce Acute de novo Lipogenesis in Human Primary Sebocytes.

Introduction: Acne arises during puberty, in part, due to elevated hormones and growth factors which stimulate de novo lipogenesis (DNL) in primary sebocytes to significantly increase sebum production. Oral isotretinoin is an effective acne therapy, reducing sebum production through inducing apoptosis in sebocytes. However, isotretinoin is teratogenic and has additional unwanted side effects, including an initial acne flare-up, which limits its utility.

Characterization of a primary brown adipocyte culture system derived from human fetal interscapular fat.

Brown fat has gained widespread attention as a potential therapeutic target to treat obesity and associated metabolic disorders. Indeed, the anti-obesity potential of multiple targets to stimulate both brown adipocyte differentiation and recruitment have been verified in rodent models. However, their therapeutic potential in humans is unknown due to the lack of a human primary brown adipocyte cell culture system.

High-content screening of human primary muscle satellite cells for new therapies for muscular atrophy/dystrophy.

Myoblast proliferation and differentiation are essential for normal skeletal muscle growth and repair. Muscle recovery is dependent on the quiescent population of muscle stem cells - satellite cells. During muscle injury, satellite cells become mitotically active and begin the repair process by fusing with each other and/or with myofibers.

Isolation and characterization of human adipose-derived stem cells for use in tissue engineering.

Human adipose-derived adult stem cells (ASCs) represent a unique population of multipotent stem cells. Their utility in a variety of tissue engineering applications, and as a model system for the study of molecular mechanisms of differentiation, is well established. In addition, their relative abundance, ease of isolation from human subcutaneous lipoaspirates, and functional stability make them an excellent physiologically relevant platform.

Differential phosphorylation of perilipin 1A at the initiation of lipolysis revealed by novel monoclonal antibodies and high content analysis.

Lipolysis in adipocytes is regulated by phosphorylation of lipid droplet-associated proteins, including perilipin 1A and hormone-sensitive lipase (HSL). Perilipin 1A is potentially phosphorylated by cAMP(adenosine 3',5'-cyclic monophosphate)-dependent protein kinase (PKA) on several sites, including conserved C-terminal residues, serine 497 (PKA-site 5) and serine 522 (PKA-site 6). To characterize perilipin 1A phosphorylation, novel monoclonal antibodies were developed, which selectively recognize perilipin 1A phosphorylation at PKA-site 5 and PKA-site 6.

Feed-forward inhibition of androgen receptor activity by glucocorticoid action in human adipocytes.

We compared transcriptomes of terminally differentiated mouse 3T3-L1 and human adipocytes to identify cell-specific differences. Gene expression and high content analysis (HCA) data identified the androgen receptor (AR) as both expressed and functional, exclusively during early human adipocyte differentiation. The AR agonist dihydrotestosterone (DHT) inhibited human adipocyte maturation by downregulation of adipocyte marker genes, but not in 3T3-L1.

EGF receptor (ERBB1) abundance in adipose tissue is reduced in insulin-resistant and type 2 diabetic women.

Context: Indications of adipose tissue dysfunction correlate with systemic insulin resistance and type 2 diabetes. It has been suggested that a defect in adipose tissue turnover may be involved in the development of these disorders. Whether this dysfunction causes or exacerbates systemic insulin resistance is not fully understood.

Metabolic remodeling of human skeletal myocytes by cocultured adipocytes depends on the lipolytic state of the system.

Objective: Adipocyte infiltration of the musculoskeletal system is well recognized as a hallmark of aging, obesity, and type 2 diabetes. Intermuscular adipocytes might serve as a benign storage site for surplus lipid or play a role in disrupting energy homeostasis as a result of dysregulated lipolysis or secretion of proinflammatory cytokines. This investigation sought to understand the net impact of local adipocytes on skeletal myocyte metabolism.

Tools for the identification of bioactives impacting the metabolic syndrome: screening of a botanical extract library using subcutaneous and visceral human adipose-derived stem cell-based assays.

Plant extracts continue to represent an untapped source of renewable therapeutic compounds for the treatment and prevention of illnesses including chronic metabolic disorders. With the increase in worldwide obesity and its related morbidities, the need for identifying safe and effective treatments is also rising. As such, use of primary human adipose-derived stem cells represents a physiologically relevant cell system to screen for bioactive agents in the prevention and treatment of obesity and its related complications.

Homeostatic levels of SRC-2 and SRC-3 promote early human adipogenesis.

The related coactivators SRC-2 and SRC-3 interact with peroxisome proliferator activated receptor γ (PPARγ) to coordinate transcriptional circuits to promote adipogenesis. To identify potential coactivator redundancy during human adipogenesis at single cell resolution, we used high content analysis to quantify links between PPARγ, SRC-2, SRC-3, and lipogenesis. Because we detected robust increases and significant cell-cell heterogeneity in PPARγ and lipogenesis, without changes in SRC-2 or SRC-3, we hypothesized that permissive coregulator levels comprise a necessary adipogenic equilibrium.

Quantification of hormone sensitive lipase phosphorylation and colocalization with lipid droplets in murine 3T3L1 and human subcutaneous adipocytes via automated digital microscopy and high-content analysis.

Lipolysis in adipocytes is associated with phosphorylation of hormone sensitive lipase (HSL) and translocation of HSL to lipid droplets. In this study, adipocytes were cultured in a high-throughput format (96-well dishes), exposed to lipolytic agents, and then fixed and labeled for nuclei, lipid droplets, and HSL (or HSL phosphorylated on serine 660 [pHSLser660]). The cells were imaged via automated digital fluorescence microscopy, and high-content analysis (HCA) methods were used to quantify HSL phosphorylation and the degree to which HSL (or pHSLser660) colocalizes with the lipid droplets.

Use of adipose-derived stem cells in high-throughput screening to identify modulators of lipogenesis.

Drug discovery efforts have an increasing focus on functional cell-based screening to identify compounds that modulate targets presented in a relevant format. Historically, immortalized cell lines have been used in primary and secondary screens due to their ease of manipulation, transformation, and propagation. However, more researchers are using primary cells that present their drug targets in their natural context.

Adipogenic differentiation of adipose-derived stem cells.

The primary physiological function of adipose-derived stem cells (ASCs) is to differentiate into adipose tissue. It is now possible to isolate, expand, and cryopreserve ASC from adipose depots of many animal species. These ASC can be induced to undergo adipogenic differentiation in vitro by exposure to a cocktail of chemical agents or inductive growth factors.

Quantification of lipid droplets and associated proteins in cellular models of obesity via high-content/high-throughput microscopy and automated image analysis.

Intracellular lipid droplets are associated with a myriad of afflictions including obesity, fatty liver disease, coronary artery disease, and infectious diseases (eg, HCV and tuberculosis). To develop high-content analysis (HCA) techniques to analyze lipid droplets and associated proteins, primary human preadipocytes were plated in 96-well dishes in the presence of rosiglitazone (rosi), a PPAR-(c) agonist that promotes adipogenesis. The cells were then labeled for nuclei, lipid droplets, and proteins such as perilipin, protein kinase C (PKC), and hormone-sensitive lipase (HSL).

The molecular mechanisms of coactivator utilization in ligand-dependent transactivation by the androgen receptor.

Androgens drive sex differentiation, bone and muscle development, and promote growth of hormone-dependent cancers by binding the nuclear androgen receptor (AR), which recruits coactivators to responsive genes. Most nuclear receptors recruit steroid receptor coactivators (SRCs) to their ligand binding domain (LBD) using a leucine-rich motif (LXXLL). AR is believed to recruit unique coactivators to its LBD using an aromatic-rich motif (FXXLF) while recruiting SRCs to its N-terminal domain (NTD) through an alternate mechanism.

Recognition and accommodation at the androgen receptor coactivator binding interface.

Prostate cancer is a leading killer of men in the industrialized world. Underlying this disease is the aberrant action of the androgen receptor (AR). AR is distinguished from other nuclear receptors in that after hormone binding, it preferentially responds to a specialized set of coactivators bearing aromatic-rich motifs, while responding poorly to coactivators bearing the leucine-rich "NR box" motifs favored by other nuclear receptors.