Although increased bone marrow fat in age‐related bone loss has been associated with lower trabecular mass, the underlying mechanism responsible remains unknown. We hypothesized that marrow ...adipocytes exert a lipotoxic effect on osteoblast function and survival through the reversible biosynthesis of fatty acids (FA) into the bone marrow microenvironment. We have used a two‐chamber system to co‐culture normal human osteoblasts (NHOst) with differentiating pre‐adipocytes in the absence or presence of an inhibitor of FA synthase (cerulenin) and separated by an insert that allowed unidirectional trafficking of soluble factors only and prevented direct cell–cell contact. Supernatants were assayed for the presence of FA using mass spectophotometry. After 3 weeks in co‐culture, NHOst showed significantly lower levels of differentiation and function based on lower mineralization and expression of alkaline phosphatase, osterix, osteocalcin and Runx2. In addition, NHOst survival was affected by the presence of adipocytes as determined by MTS‐formazan and TUNEL assays as well as higher activation of caspases 3/7. These toxic effects were inhibited by addition of cerulenin. Furthermore, culture of NHOst with either adipocyte‐conditioned media alone in the absence of adipocytes themselves or with the addition of the most predominant FA (stearate or palmitate) produced similar toxic results. Finally, Runx2 nuclear binding was affected by addition of either adipocyte conditioned media or FA into the osteogenic media. We conclude that the presence of FA within the marrow milieu can contribute to the age‐related changes in bone mass and can be prevented by the inhibition of FA synthase.
Adipose tissue is now recognized as an accessible, abundant, and reliable site for the isolation of adult stem cells suitable for tissue engineering and regenerative medicine applications. The past ...decade has witnessed an explosion of preclinical data relating to the isolation, characterization, cryopreservation, differentiation, and transplantation of freshly isolated stromal vascular fraction cells and adherent, culture-expanded, adipose-derived stromal/stem cells in vitro and in animal models. This body of work has provided evidence supporting clinical translational applications of adipose-derived cells in safety and efficacy trials. The present article reviews the case reports and phase I-III clinical evidence using autologous adipose-derived cells that have been published, to date, in the fields of gastroenterology, neurology, orthopedics, reconstructive surgery, and related clinical disciplines. Future directions and challenges facing the field are discussed and evaluated.
We report the generation and comparative analysis of genome-wide chromatin state maps, PPARγ and CTCF localization maps, and gene expression profiles from murine and human models of adipogenesis. The ...data provide high-resolution views of chromatin remodeling during cellular differentiation and allow identification of thousands of putative preadipocyte- and adipocyte-specific cis-regulatory elements based on dynamic chromatin signatures. We find that the specific locations of most such elements differ between the two models, including at orthologous loci with similar expression patterns. Based on sequence analysis and reporter assays, we show that these differences are determined, in part, by evolutionary turnover of transcription factor motifs in the genome sequences and that this turnover may be facilitated by the presence of multiple distal regulatory elements at adipogenesis-dependent loci. We also utilize the close relationship between open chromatin marks and transcription factor motifs to identify and validate PLZF and SRF as regulators of adipogenesis.
Display omitted
► Chromatin state maps were generated throughout murine and human adipogenesis ► Most active cis-regulatory elements differ between species ► Many differences are due to evolutionary turnover of transcription factor motifs ► Motif enrichment predicted that PLZF and SRF play a repressive role in adipogenesis
Emerging evidence suggests that increases in activated T cell populations in adipose tissue may contribute toward obesity-associated metabolic syndrome. The present study investigates three ...unanswered questions: 1) Do adipose-resident T cells (ARTs) from lean and obese mice have altered cytokine production in response to TCR ligation?; 2) Do the extralymphoid ARTs possess a unique TCR repertoire compared with lymphoid-resident T cells and whether obesity alters the TCR diversity in specific adipose depots?; and 3) Does short-term elimination of T cells in epididymal fat pad without disturbing the systemic T cell homeostasis regulate inflammation and insulin-action during obesity? We found that obesity reduced the frequency of naive ART cells in s.c. fat and increased the effector-memory populations in visceral fat. The ARTs from diet-induced obese (DIO) mice had a higher frequency of IFN-gamma(+), granzyme B(+) cells, and upon TCR ligation, the ARTs from DIO mice produced increased levels of proinflammatory mediators. Importantly, compared with splenic T cells, ARTs exhibited markedly restricted TCR diversity, which was further compromised by obesity. Acute depletion of T cells from epididymal fat pads improved insulin action in young DIO mice but did not reverse obesity-associated feed forward cascade of chronic systemic inflammation and insulin resistance in middle-aged DIO mice. Collectively, these data establish that ARTs have a restricted TCR-Vbeta repertoire, and T cells contribute toward the complex proinflammatory microenvironment of adipose tissue in obesity. Development of future long-term T cell depletion protocols specific to visceral fat may represent an additional strategy to manage obesity-associated comorbidities.
Abstract Delivery systems providing spatial and temporal control have the potential to improve outcomes in surgical reconstruction and regenerative medicine by precise modulation of wound healing and ...tissue repair processes. In this study we describe a synthesis and oligonucleotide functionalization process of silver nanoparticle complexes for photoactivated microRNA (miRNA) delivery. The activity of the PC-miR-148b–SNP construct is demonstrated by light mediated delivery of miR-148b mimic resulting in differentiation of human autologous adipose derived mesenchymal stromal/stem cells (hASCs) into an osteogenic linage. The conjugate, upon photoactivation, increases alkaline phosphatase (ALP) activity in the cell membrane and calcification (mineralization) of hASCs on days 7 and 14 respectively. Additionally, the expression of mRNA for the early, middle and late stage osteogenic markers; ALP, RunX2 and osteocalcin (OCN) respectively, was also significantly upregulated at days 7 and 28, respectively after photoactivation of PC-miR-148b–SNP and release of miR-148b mimics. Additionally, PC-miR-148b–SNP conjugate is readily delivered to the intracellular compartment without the use of transfection vectors commonly required for free oligonucleotides. This technology demonstrates photo-controlled, spatial and temporal modulation of osteogenesis in hASCs.
The pace of discovery involving adipose-derived cells continues to accelerate at both the preclinical and clinical translational levels. Adipose tissue is a source of freshly isolated, heterogeneous ...stromal vascular fraction cells and culture-expanded, adherent and relatively homogeneous adipose stromal/stem cells. Both populations display regenerative capacity in soft and hard tissue repair, ischemic insults and autoimmune diseases. While their major mechanism of action has been attributed to both direct lineage differentiation and/or paracrine factor release, current evidence favors a paracrine mechanism. Over 40 clinical trials using adipose-derived cells conducted in 15 countries have been registered with the NIH, the majority of which are Phase I or Phase I/II safety studies. This review focuses on the literature of the past 2 years in order to assess the status of clinical and preclinical studies on adipose-derived cell therapies for regenerative medicine.
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.
Summary
The regeneration potential of mesenchymal stem cells (MSCs) diminishes with advanced age and this diminished potential is associated with changes in cellular functions. This study compared ...MSCs isolated from the bone marrow of rhesus monkeys (rBMSCs) in three age groups: young (< 5 years), middle (8–10 years), and old (> 12 years). The effects of aging on stem cell properties and indicators of stem cell fitness such as proliferation, differentiation, circadian rhythms, stress response proteins, miRNA expression, and global histone modifications in rBMSCs were analyzed. rBMSCs demonstrated decreased capacities for proliferation and differentiation as a function of age. The production of heat shock protein 70 (HSP70) and heat shock factor 1 (HSF1) were also reduced with increasing age. The level of a core circadian protein, Rev‐erb α, was significantly increased in rBMSCs from old animals. Furthermore, analysis of miRNA expression profiles revealed an up‐regulation of mir‐766 and mir‐558 and a down‐regulation of mir‐let‐7f, mir‐125b, mir‐222, mir‐199‐3p, mir‐23a, and mir‐221 in old rBMSCs compare to young rBMSCs. However, there were no significant age‐related changes in the global histone modification profiles of the four histone core proteins: H2A, H2B, H3, and H4 on rBMSCs. These changes represent novel insights into the aging process and could have implications regarding the potential for autologous stem cells therapy in older patients.
PDF
