Previous studies have demonstrated that EGF and bFGF maintain the stem cell properties of proliferating human adipose-derived stromal/stem cells (hASCs) in vitro. While the expansion and cryogenic preservation of isolated hASCs are routine, these manipulations can impact their proliferative and differentiation potential. This study examined cryogenically preserved hASCs (n = 4 donors), with respect to these functions, after culture with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) at varying concentrations (0–10 ng/ml). Relative to the control, cells supplemented with EGF and bFGF significantly increased proliferation by up to three-fold over 7–8 days. Furthermore, cryopreserved hASCs expanded in the presence of EGF and bFGF displayed increased oil red O staining following adipogenic induction. This was accompanied by significantly increased levels of several adipogenesis-related mRNAs: aP2, C/EBPα, lipoprotein lipase (LPL), PPARγ and PPARγ co-activator-1 (PGC1). Adipocytes derived from EGF- and bFGF-cultured hASCs exhibited more robust functionality based on insulin-stimulated glucose uptake and atrial natriuretic peptide (ANP)-stimulated lipolysis. These findings indicate that bFGF and EGF can be used as culture supplements to optimize the proliferative capacity of cryopreserved human ASCs and their adipogenic differentiation potential.

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. The current chapter describes methods to induce adipogenesis and to quantify this differentiation process in vitro.

A core group of regulatory factors control circadian rhythms in mammalian cells. While the suprachiasmatic nucleus in the brain serves as the central core circadian oscillator, circadian clocks also exist within peripheral tissues and cells. A growing body of evidence has demonstrated that >20% of expressed mRNAs in bone and adipose tissues oscillate in a circadian manner. The current manuscript reports evidence of the core circadian transcriptional apparatus within primary cultures of murine and human bone marrow-derived mesenchymal stem cells (BMSCs). Exposure of confluent, quiescent BMSCs to dexamethasone synchronized the oscillating expression of the mRNAs encoding the albumin D binding protein (dbp), brain-muscle arnt-like 1 (bmal1), period 3 (per3), rev-erb alpha (Rev A), and rev-erb beta (Rev B). The genes displayed a mean oscillatory period of 22.2 to 24.3 h. The acrophase or peak expression of mRNAs encoding "positive" (bmal1) and "negative" (per3) components of the circadian regulatory apparatus were out of phase with each other by approximately 8-12 h, consistent with in vivo observations. In vivo, phosphyrylation by glycogen synthase kinase 3beta (GSK3beta) is known to regulate the turnover of per3 and components of the core circadian regulatory apparatus. In vitro addition of lithium chloride, a GSK3beta inhibitor, significantly shifted the acrophase of all genes by 4.2-4.7 h oscillation in BMSCs; however, only the male murine BMSCs displayed a significant increase in the length of the period of oscillation. We conclude that human and murine BMSCs represent a valid in vitro model for the analysis of circadian mechanisms in bone metabolism and stem cell biology.

Objective Circadian mechanisms underlie the physiology of mammals as an adaptation to the earth’s rotation on its axis. Highly conserved core circadian regulatory proteins (CCRP) maintain an oscillatory expression profile in the central and peripheral tissues. The CCRP include both a positive and negative arm as well as downstream transcriptional regulators. Recent studies in murine models have determined that the mRNAs encoding the CCRP are present in multiple adipose tissue depots and exhibit a robust oscillatory expression profile. The current study set out to examine the expression of CCRP mRNAs in human subcutaneous adipose tissues. Design Retrospective analysis of total RNA isolated from subcutaneous adipose tissue. Subjects 150 healthy female and male lean (BMI < 25), overweight (BMI between 25 and 29.99) or obese (BMI >30) subjects of varied ethnic backgrounds undergoing elective liposuction or surgical procedures. Results The expression of the CCRP mRNAs displayed a significant correlation between each other and mRNAs representative of adipogenic biomarkers. Hierarchical cluster analyses of mRNAs isolated from the cohort of female Caucasian subjects (n = 116) identified three major clusters based on expression of downstream CCRP mRNAs. The mRNAs encoding D site of albumin promoter binding protein (DBP), E4 promoter binding protein 4 (E4BP4), PPARγ Co-Activator 1β (PGC-1β), and Rev-erb α were negatively correlated with BMI in a lean cluster (n = 66), positively correlated with BMI in a younger overweight/obese cluster (n = 19), and not significantly correlated with BMI in an older, overweight/obese cluster (n = 31). Conclusions These data confirm and extend findings that link the CCRP and circadian mechanisms to the risk of obesity.