Isolation of human adipose-derived stem cells from biopsies and liposuction specimens.

Adipose tissue has proven to serve as an abundant, accessible, and rich source of adult stem cells with multipotent properties suitable for tissue engineering and regenerative medical applications. Here, we describe a detailed method for the isolation and expansion of adipose-derived stem cells (ASCs). We present a large scale procedure suitable for processing >100 mL volumes of lipoaspirate tissue specimens and a small scale procedure suitable for processing adipose tissue biopsy specimens of < 0.

Induction of circadian gene expression in human subcutaneous adipose-derived stem cells.

Objective: Genes encoding the circadian transcriptional apparatus exhibit robust oscillatory expression in murine adipose tissues. This study tests the hypothesis that human subcutaneous adipose-derived stem cells (ASCs) provide an in vitro model in which to monitor the activity of the core circadian transcriptional apparatus.

Research Methods And Procedures: Primary cultures of undifferentiated or adipocyte-differentiated ASCs were treated with dexamethasone, rosiglitazone, or 30% fetal bovine serum.

Digital signal processing reveals circadian baseline oscillation in majority of mammalian genes.

In mammals, circadian periodicity has been described for gene expression in the hypothalamus and multiple peripheral tissues. It is accepted that 10%-15% of all genes oscillate in a daily rhythm, regulated by an intrinsic molecular clock. Statistical analyses of periodicity are limited by the small size of datasets and high levels of stochastic noise.

Circadian rhythms and the regulation of metabolic tissue function and energy homeostasis.

Circadian oscillators play an indispensable role in the coordination of physiological processes with the cyclic changes in the physical environment. A significant number of recent clinical and molecular studies suggest that circadian biology may play an important role in the regulation of adipose and other metabolic tissue functions. In this discussion, we present the hypothesis that circadian dysfunction may be involved in the pathogenesis of obesity, type 2 diabetes, and the metabolic syndrome.

Fine-Tuning Reception in the Bone: PPARgamma and Company.

PPARgamma plays a central role in the formation of fat. Regulation of PPARgamma activity depends on numerous factors ranging from dietary ligands to nuclear hormone coactivators and corepressors to oxygen-sensing mechanisms. In addition, the interplay of PPARgamma with other nuclear hormone receptors has implications for the balance between adipogenesis and osteogenesis in mesenchymal stem cells of the bone marrow stroma.

Circadian oscillation of gene expression in murine calvarial bone.

Unlabelled: The genes encoding the core circadian transcription factors display an oscillating expression profile in murine calvarial bone. More than 26% of the calvarial bone transcriptome exhibits a circadian rhythm, comparable with that observed in brown and white adipose tissues and liver. Thus, circadian mechanisms may directly modulate oxidative phosphorylation and multiple metabolic pathways in bone homeostasis.

Permutation test for periodicity in short time series data.

Background: Periodic processes, such as the circadian rhythm, are important factors modulating and coordinating transcription of genes governing key metabolic pathways. Theoretically, even small fluctuations in the orchestration of circadian gene expression patterns among different tissues may result in functional asynchrony, at the organism level, and may contribute to a wide range of pathologic disorders. Identification of circadian expression pattern in time series data is important, but equally challenging.

Secretome of primary cultures of human adipose-derived stem cells: modulation of serpins by adipogenesis.

Studies of adipogenic protein induction have led to a new appreciation of the role of adipose tissue as an endocrine organ. Adipocyte-derived "adipokines" such as adiponectin, leptin, and visceral adipose tissue-derived serine protease inhibitor (vaspin) exert hormone-like activities at the systemic level. In this study, we examined the secretome of primary cultures of human subcutaneous adipose-derived stem cells as an in vitro model of adipogenesis.

Characterization of peripheral circadian clocks in adipose tissues.

First described in the suprachiasmatic nucleus, circadian clocks have since been found in several peripheral tissues. Although obesity has been associated with dysregulated circadian expression profiles of leptin, adiponectin, and other fat-derived cytokines, there have been no comprehensive analyses of the circadian clock machinery in adipose depots. In this study, we show robust and coordinated expression of circadian oscillator genes (Npas2, Bmal1, Per1-3, and Cry1-2) and clock-controlled downstream genes (Rev-erb alpha, Rev-erb beta, Dbp, E4bp4, Stra13, and Id2) in murine brown, inguinal, and epididymal (BAT, iWAT, and eWAT) adipose tissues.

Circadian clocks are resounding in peripheral tissues.

Circadian rhythms are prevalent in most organisms. Even the smallest disturbances in the orchestration of circadian gene expression patterns among different tissues can result in functional asynchrony, at the organism level, and may to contribute to a wide range of physiologic disorders. It has been reported that as many as 5%-10% of transcribed genes in peripheral tissues follow a circadian expression pattern.

Playing with bone and fat.

The relationship between bone and fat formation within the bone marrow microenvironment is complex and remains an area of active investigation. Classical in vitro and in vivo studies strongly support an inverse relationship between the commitment of bone marrow-derived mesenchymal stem cells or stromal cells to the adipocyte and osteoblast lineage pathways. In this review, we focus on the recent literature exploring the mechanisms underlying these differentiation events and discuss their implications relevant to osteoporosis and regenerative medicine.

The immunogenicity of human adipose-derived cells: temporal changes in vitro.

Regenerative medical techniques will require an abundant source of human adult stem cells that can be readily available at the point of care. The ability to use unmatched allogeneic stem cells will help achieve this goal. Since adipose tissue represents an untapped reservoir of human cells, we have compared the immunogenic properties of freshly isolated, collagenase-digested human adipose tissue-derived stromal vascular fraction cells (SVFs) relative to passaged, plastic-adherent adipose-derived stem cells (ASCs).

Immunophenotype of human adipose-derived cells: temporal changes in stromal-associated and stem cell-associated markers.

Adipose tissue represents an abundant and accessible source of multipotent adult stem cells and is used by many investigators for tissue engineering applications; however, not all laboratories use cells at equivalent stages of isolation and passage. We have compared the immunophenotype of freshly isolated human adipose tissue-derived stromal vascular fraction (SVF) cells relative to serial-passaged adipose-derived stem cells (ASCs). The initial SVF cells contained colony-forming unit fibroblasts at a frequency of 1:32.

Effect of various freezing parameters on the immediate post-thaw membrane integrity of adipose tissue derived adult stem cells.

The effect of four thermal parameters on post-thaw membrane integrity of adipose tissue derived adult stem (ADAS) cells after controlled-rate freezing was investigated with the help of a two-level four-parameter (2(4)) experimental design. The four thermal parameters studied were cooling rate (CR), end temperature (ET), hold time (HT), and thawing rate (TR). Several passages, including Passage-0 (P0), Passage-1 (P1), Passage-2 (P2), Passage-3 (P3), and Passage-4 (P4), obtained from the suspended culture of stromal vascular fraction (SVF) of the ADAS cells were used for this study.

Cross-talk among gp130 cytokines in adipocytes.

The interleukin-6 (IL-6) family of cytokines is a family of structurally and functionally related proteins, including IL-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), and cardiotrophin-1 (CT-1). These proteins are also known as gp130 cytokines because they all share gp130 as a common transducer protein within their functional receptor complexes. Several of these cytokines (LIF, OSM, CNTF, and CT-1) also utilize the LIF receptor (LIFR) as a component of their receptor complex.

Proteomic analysis of primary cultures of human adipose-derived stem cells: modulation by Adipogenesis.

Adipogenesis plays a critical role in energy metabolism and is a contributing factor to the obesity epidemic. This study examined the proteome of primary cultures of human adipose-derived adult stem (ADAS) cells as an in vitro model of adipogenesis. Protein lysates obtained from four individual donors were compared before and after adipocyte differentiation by two-dimensional gel electrophoresis and tandem mass spectroscopy.

Effects of cardiotrophin on adipocytes.

Cardiotrophin (CT-1) is a naturally occurring protein member of the interleukin (IL)-6 cytokine family and signals through the gp130/leukemia inhibitory factor receptor (LIFR) heterodimer. The formation of gp130/LIFR complex triggers the auto/trans-phosphorylation of associated Janus kinases, leading to the activation of Janus kinase/STAT and MAPK (ERK1 and -2) signaling pathways. Since adipocytes express both gp130 and LIFR proteins and are responsive to other IL-6 family cytokines, we examined the effects of CT-1 on 3T3-L1 adipocytes.

Growth hormone, but not insulin, activates STAT5 proteins in adipocytes in vitro and in vivo.

STAT 5 proteins are latent transcription factors which have been shown to be activated by growth hormone (GH) in many cell types. However, some recent studies also suggest that STAT 5B is a physiological substrate of the insulin receptor. In our studies, we have shown that physiological levels of insulin do not induce STAT 5 tyrosine phosphorylation or affect the nuclear distribution of STATs 5A or 5B in 3T3-L1 adipocytes.

The regulation and activation of ciliary neurotrophic factor signaling proteins in adipocytes.

Ciliary neurotrophic factor (CNTF) is primarily known for its roles as a lesion factor released by the ruptured glial cells that prevent neuronal degeneration. However, CNTF has also been shown to cause weight loss in a variety of rodent models of obesity/type II diabetes, whereas a modified form also causes weight loss in humans. CNTF administration can correct or improve hyperinsulinemia, hyperphagia, and hyperlipidemia associated with these models of obesity.