Sanjin Zvonic

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. Colony‐forming unit adipocytes and osteoblasts were present in the SVF cells at comparable frequencies (1:28 and 1:16, respectively). The immunophenotype of the adipose‐derived cells based on flow cytometry changed progressively with adherence and passage. Stromal cell–associated markers (CD13, CD29, CD44, CD63, CD73, CD90, CD166) were initially low on SVF cells and increased significantly with successive passages. The stem cell–associated marker CD34 was at peak levels in the SVF cells and/or early‐passage ASCs and remained present, although at reduced levels, throughout the culture period. Aldehyde dehydrogenase and the multidrug‐resistance transport protein (ABCG2), both of which have been used to identify and characterize hematopoietic stem cells, are expressed by SVF cells and ASCs at detectable levels. Endothelial cell–associated markers (CD31, CD144 or VE‐cadherin, vascular endothelial growth factor receptor 2, von Willebrand factor) were expressed on SVF cells and did not change significantly with serial passage. Thus, the adherence to plastic and subsequent expansion of human adipose‐derived cells in fetal bovine serum‐supplemented medium selects for a relatively homogeneous cell population, enriching for cells expressing a stromal immunophenotype, compared with the heterogeneity of the crude SVF.

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 adipoctye 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. J. Cell. Biochem. 98: 251–266, 2006.

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). Parallel studies have shown that adherence to plastic and subsequent expansion of human adipose‐derived cells selects for a relatively homogeneous cell population based on immunophenotype. Consistent with these findings, the presence of hematopoietic‐associated markers (CD11a, CD14, CD45, CD86, and histocompatible locus antigen‐DR [HLA‐DR]) detected on the heterogeneous SVF cell population decreased upon subsequent passage of the ASCs. In mixed lymphocyte reactions (MLRs), SVFs, and early passage ASCs stimulated proliferation by allogeneic responder T cells. In contrast, the ASCs beyond passage P1 failed to elicit a response from T cells. Indeed, late passage ASCs actually suppressed the MLR response. Although these results support the feasibility of allogeneic human ASC transplantation, confirmatory in vivo animal studies will be required.

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 < 0.5 g. Although we have focused on the isolation of ASCs from human adipose tissue, the procedure can be applied to adipose tissues from other species with minimal modifications.

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. Over 170 individual protein features in the undifferentiated adipose-derived adult stem cells were identified. Following adipogenesis, over 40 proteins were up-regulated by ≥2-fold, whereas 13 showed a ≥3-fold reduction. The majority of the modulated proteins belonged to the following functional categories: cytoskeleton, metabolic, redox, protein degradation, and heat shock protein/chaperones. Additional immunoblot analysis documented the induction of four individual heat shock proteins and confirmed the presence of the heat shock protein 27 phosphoserine 82 isoform, as predicted by the proteomic analysis, as well as the crystallin α phosphorylated isoforms. These findings suggest that the heat shock protein family proteome warrants further investigation with respect to the etiology of obesity and type 2 diabetes.

Circadian oscillation of gene expression in murine calvarial bone.

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.

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.

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. Here, we propose a new approach applying digital signal processing algorithms separately to each group of genes oscillating in the same phase. Combined with the statistical tests for periodicity, this method identifies circadian baseline oscillation in almost 100% of all expressed genes. Consequently, circadian oscillation in gene expression should be evaluated in any study related to biological pathways. Changes in gene expression caused by mutations or regulation of environmental factors (such as photic stimuli or feeding) should be considered in the context of changes in the amplitude and phase of genetic oscillations.

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.

Permutation test for periodicity in short time series data

BackgroundPeriodic 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. Microarray technology allows estimation of relative expression of thousands of genes at each time point. However, this estimation often lacks precision and microarray experiments are prohibitively expensive, limiting the number of data points in a time series expression profile. The data produced in these experiments carries a high degree of stochastic variation, obscuring the periodic pattern and a limited number of replicates, typically covering not more than two complete periods of oscillation.ResultsTo address this issue, we have developed a simple, but effective, computational technique for the identification of a periodic pattern in relatively short time series, typical for microarray studies of circadian expression. This test is based on a random permutation of time points in order to estimate non-randomness of a periodogram. The Permutated time, or Pt-test, is able to detect oscillations within a given period in expression profiles dominated by a high degree of stochastic fluctuations or oscillations of different irrelevant frequencies. We have conducted a comprehensive study of circadian expression on a large data set produced at PBRC, representing three different peripheral murine tissues. We have also re-analyzed a number of similar time series data sets produced and published independently by other research groups over the past few years.ConclusionThe Permutated time test (Pt-test) is demonstrated to be effective for detection of periodicity in short time series typical for high-density microarray experiments. The software is a set of C++ programs available from the authors on the open source basis.