Adipogenic Lineage Commitment Research Articles

Identification of novel pharmacological activators of circadian clock with anti-adipogenic properties

The circadian clock drives rhythmic oscillations of metabolic processes to orchestrate metabolic homeostasis, and disruption of this mechanism predisposes to obesity and insulin resistance. Preserving or augmenting clock function could be a potential therapeutic target for metabolic diseases, particularly with the wide-spread of circadian misalignment in a modern lifestyle. To date, targeting the clock for metabolic disease prevention or treatment remains to be explored. Adipocyte possesses cell-autonomous clocks that exert transcriptional control of the Wnt pathway to inhibit adipocyte development. Using a high through-put screening pipeline, we recently identified Chlorhexidine as a novel clock-activating molecule that targets the key driver of the circadian clock transcriptional feedback loop, the CLOCK protein. Based on clock function in suppressing adipogenesis, we hypothesize that Chlorhexidine and related compounds may possess adipogenic-inhibitory properties suitable for anti-obesity drug development. In distinct adipogenic progenitor models, we demonstrate the activity of Chlorhexidine in inhibiting adipogenic lineage commitment and terminal differentiation. Furthermore, we report the structural optimization of Chlorhexidine chemical scaffold that led to the discovery of new analogs with improved anti-adipogenic effcacy. Consistent with its activity for clock activation, in adipogenic progenitors containing a Period2::dLuc luciferase reporter, Chlorhexidine induced significant shortening of clock period length with induction of core clock components. Chlorhexidine treatment of adipogenic mesenchymal precursors robustly suppressed their adipogenic maturation, with a comparable effect observed on inhibiting terminal differentiation of primary preadipocytes in a clock-dependent manner. Mechanistically, Chlorhexidine stimulates clock-controlled Wnt signaling that mediates its anti-adipogenic effect. Through medicinal chemistry modification of its chemical scaffold, we generated a panel of Chlorhexidine analogs with validation for their clock-modulatory activities. Structure activity relationship analysis of these analogs led to the identification of CM002 as a new clock activator with improved clock-dependent anti-adipogenic activity. Collectively, our findings uncovered a new class of clock-modulatory compounds that inhibit adipocyte development for potential anti-obesity drug discovery and provide clock-targeting chemical probes for dissecting clock function in metabolic physiology. This project was supported by grants from National Institute of Aging R56AG080294 and Arthur Riggs-Diabetes & Metabolism Research Institute T2D and T1D Innovative Awards to KM. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Metformin potentiates immunosuppressant activity and adipogenic differentiation of human umbilical cord-mesenchymal stem cells

Metformin, a first-line drug for type-2 diabetes, displays pleiotropic effects on inflammation, aging, and cancer. Obesity triggers a low-grade chronic inflammation leading to insulin resistance, characterized by increased pro-inflammatory cytokines produced by adipocytes and infiltrated immune cells, which contributes to metabolic syndrome.We investigated metformin’s differentiation and immunoregulatory properties of human umbilical cord-mesenchymal stem cells (UC-MSC), as cellular basis of its beneficial role in metabolic dysfunctions.Isolation, characterization and multilineage differentiation of UC-MSC were performed using standard protocols and flow-cytometry. Metformin effects on UC-MSC growth was assessed by colony formation and MTT assay, gene and protein expression by qRT-PCR, and western blot analysis. Proliferation of peripheral blood mononuclear cells (PBMCs) co-cultured with metformin-treated UC-MSC-conditioned media was evaluated by dye dilution assay.We show that metformin decreases proliferation and colony formation of UC-MSCs and enhances their adipogenic lineage commitment. Metformin (3 mM) increases PPARγ and downregulates FABP4 mRNA both in basal and in adipogenic culture conditions; however, the modulation of PPARγ expression is unrelated to the antiproliferative effects. Moreover, metformin inhibits UC-MSC inflammatory activity reducing the expression of IL-6, MCP-1, and COX-2. Conditioned media, collected from metformin-treated UC-MSCs, down-regulate CD3+ T lymphocyte growth in stimulated PBMCs and, in particular, reduce the CD8+ T cell population.These results indicate that metformin may favor new adipocyte formation and potentiate immune suppressive properties of UC-MSCs. Thus, adipose tissue regeneration and anti-inflammatory activity may represent possible mechanisms by which metformin exerts its positive effect on lipid metabolism.

Autophagy perturbation upon acute pyrethroid treatment impacts adipogenic commitment of mesenchymal stem cells

Environmental chemical exposure can cause dysregulation in adipogenesis that can result in metabolic syndrome, which includes insulin resistance, type 2 diabetes, cardiovascular disease, as well as excessive body weight. The role of autophagy in adipocyte differentiation is debatable since both positive and negative effects have been reported. Type-I and type-II synthetic pyrethroids α-cypermethrin (CPM) and permethrin (PER), respectively, are reported to increase adipogenesis in vitro and in vivo. However, it is not known how these pyrethroids affect mesenchymal stem cells (MSCs). Thus, this study focused on evaluating the effect of pyrethroids (CPM and PER) pre-treatment (24 h) on MSC commitment and the regulatory role of autophagy in adipogenic lineage commitment. The formation of adipocytes was observed through nile red staining, perilipin expression by immunoflourescence, and adipogenic markers PPARγ, C/EBPα, and FABP4 by western blotting. It was found that the adipogenic differentiation ability of MSCs was significantly increased upon CPM or PER pre-treatment at 100 μM concentration as evident by lipid accumulation and enhanced expression of adipogenic markers. To assess the involvement of autophagy, the expression of p62 and LC3II were evaluated following pre-treatment. Immunoblotting results revealed an increased expression of p62 and LC3II in CPM or PER pretreated MSCs suggesting CPM and PER mediated inhibition of autophagy at 24 h. Further, an increase was observed in adipogenesis upon CPM or PER pre-treatment in combination with chloroquine, while use of rapamycin during pre-treatment abrogated the effect of CPM and PER. Thus, this study concludes that CPM or PER pre-treatment increases the adipogenic differentiation of MSCs. Since chloroquine also demonstrated similar adipogenic response, it further highlights that 24 h pre-treatment with autophagy modulators to inhibit basal autophagy primes MSCs towards adipogenic lineage.

Distinct Adipogenic and Fibrogenic Differentiation Capacities of Mesenchymal Stromal Cells from Pancreas and White Adipose Tissue.

Pancreatic steatosis associates with β-cell failure and may participate in the development of type-2-diabetes. Our previous studies have shown that diabetes-susceptible mice accumulate more adipocytes in the pancreas than diabetes-resistant mice. In addition, we have demonstrated that the co-culture of pancreatic islets and adipocytes affect insulin secretion. The aim of this current study was to elucidate if and to what extent pancreas-resident mesenchymal stromal cells (MSCs) with adipogenic progenitor potential differ from the corresponding stromal-type cells of the inguinal white adipose tissue (iWAT). miRNA (miRNome) and mRNA expression (transcriptome) analyses of MSCs isolated by flow cytometry of both tissues revealed 121 differentially expressed miRNAs and 1227 differentially expressed genes (DEGs). Target prediction analysis estimated 510 DEGs to be regulated by 58 differentially expressed miRNAs. Pathway analyses of DEGs and miRNA target genes showed unique transcriptional and miRNA signatures in pancreas (pMSCs) and iWAT MSCs (iwatMSCs), for instance fibrogenic and adipogenic differentiation, respectively. Accordingly, iwatMSCs revealed a higher adipogenic lineage commitment, whereas pMSCs showed an elevated fibrogenesis. As a low degree of adipogenesis was also observed in pMSCs of diabetes-susceptible mice, we conclude that the development of pancreatic steatosis has to be induced by other factors not related to cell-autonomous transcriptomic changes and miRNA-based signals.

Functional expression of ZNF467 and PCBP2 supports adipogenic lineage commitment in adipose-derived mesenchymal stem cells

Bone marrow-derived mesenchymal stromal/stem cells (BMSCs) have the potential to be employed in many different skeletal therapies. A major limitation to utilizing BMSCs as a therapeutic strategy in human disease and tissue regeneration is the low cell numbers obtained from initial isolation necessitating multiple cell passages that can lead to decreased cell quality. Adipose-derived mesenchymal stromal/stem cells (AMSCs) have been proposed as an alternative cell source for regenerative therapies; however the differentiation capacity of these cells differs from BMSCs. To understand the differences between BMSCs and AMSCs, we compared the global gene expression profiles of BMSCs and AMSCs and identified two genes, PCBP2 and ZNF467 that were differentially expressed between AMSCs and BMSCs. We demonstrate that PCBP2 and ZNF467 impact adipogenic but not osteogenic differentiation, further supporting evidence that AMSCs and BMSCs appear to be adapted to their microenvironment.

Vulpinic Acid Controls Stem Cell Fate toward Osteogenesis and Adipogenesis.

Vulpinic acid, a naturally occurring methyl ester of pulvinic acid, has been reported to exert anti-fungal, anti-cancer, and anti-oxidative effects. However, its metabolic action has not been implicated yet. Here, we show that vulpinic acid derived from a mushroom, Pulveroboletus ravenelii controls the cell fate of mesenchymal stem cells and preadipocytes by inducing the acetylation of histone H3 and α-tubulin, respectively. The treatment of 10T1/2 mesenchymal stem cells with vulpinic acid increased the expression of Wnt6, Wnt10a, and Wnt10b, which led to osteogenesis inhibiting the adipogenic lineage commitment, through the upregulation of H3 acetylation. By contrast, treatment with vulpinic acid promoted the terminal differentiation of 3T3-L1 preadipocytes into mature adipocytes. In this process, the increase in acetylated tubulin was accompanied, while acetylated H3 was not altered. As excessive generation of adipocytes occurs, the accumulation of lipid drops was not concentrated, but dispersed into a number of adipocytes. Consistently, the expressions of lipolytic genes were upregulated and inflammatory factors were downregulated in adipocytes exposed to vulpinic acid during adipogenesis. These findings reveal the multiple actions of vulpinic acid in two stages of differentiation, promoting the osteogenesis of mesenchymal stem cells and decreasing hypertrophic adipocytes, which can provide experimental evidence for the novel metabolic advantages of vulpinic acid.

Growth hormone facilitates 5′-azacytidine-induced myogenic but inhibits 5′-azacytidine-induced adipogenic commitment in C3H10T1/2 mesenchymal stem cells

The C3H10T1/2 cells are considered mesenchymal stem cells (MSCs) because they can be induced to become the progenitor cells for myocytes, adipocytes, osteoblasts, and chondrocytes by the DNA methyltransferase inhibitor 5′-azacytidine. In this study, we determined the effect of growth hormone (GH) on the myogenic and adipogenic lineage commitment in C3H10T1/2 cells. The C3H10T1/2 cells were treated with recombinant bovine GH in the presence or absence of 5′-azacytidine for 4 days. The myogenic commitment in C3H10T1/2 cells was assessed by immunostaining them for MyoD, the marker for myoblasts, and by determining their capacity to differentiate into the multinucleated myotubes. The adipogenic commitment in C3H10T1/2 cells was assessed by determining their ability to differentiate into adipocytes. Myotubes and adipocyteswere identified by immunocytochemistry and Oil Red O staining, respectively. C3H10T1/2 cells treated with 5′-azacytidine and GH for 4 days contained a greater percentage of MyoD-positive cells than those treated with 5′-axacytidine alone (P < 0.05). The former generated more myotubes than the latter upon induced myoblast differentiation (P < 0.05). However, C3H10T1/2 cells treated with GH alone did not form any myotubes. C3H10T1/2 cells treated with 5′-azacytidine formed adipocytes upon adipocyte differentiation induction, whereas C3H10T1/2 cells treated with GH alone did not form any adipocytes. C3H10T1/2 cells treated with both 5′-azacytidine and GH formed fewer adipocytes than those treated with 5′-azacytidine alone (P < 0.05). Both GHR and IGF-I mRNA expression in C3H10T1/2 cells were increased by 5′-azacytidine (P < 0.05), but neither was affected by GH. Overall, this study showed that GH enhanced 5′-azacytidine-induced commitment in C3H10T1/2 cells to myoblasts but inhibited 5′-azacytidine-induced commitment to preadipocytes. These results support the possibility that GH stimulates skeletal muscle growth and inhibits adipose tissue growth in part by stimulating the myogenic commitment and inhibiting the adipogenic commitment, respectively, in mesenchymal stem cells.

Identification of Three Early Phases of Cell-Fate Determination during Osteogenic and Adipogenic Differentiation by Transcription Factor Dynamics

SummaryAge-related skeletal degeneration in patients with osteoporosis is characterized by decreased bone mass and occurs concomitant with an increase in bone marrow adipocytes. Using microarray expression profiling with high temporal resolution, we identified gene regulatory events in early stages of osteogenic and adipogenic lineage commitment of human mesenchymal stromal cells (hMSCs). Data analysis revealed three distinct phases when cells adopt a committed expression phenotype: initiation of differentiation (0–3 hr, phase I), lineage acquisition (6–24 hr, phase II), and early lineage progression (48–96 hr, phase III). Upstream regulator analysis identified 34 transcription factors (TFs) in phase I with a role in hMSC differentiation. Interestingly, expression levels of identified TFs did not always change and indicate additional post-transcriptional regulatory mechanisms. Functional analysis revealed that forced expression of IRF2 enhances osteogenic differentiation. Thus, IRF2 and other early-responder TFs may control osteogenic cell fate of MSCs and should be considered in mechanistic models that clarify bone-anabolic changes during clinical progression of osteoporosis.

MiR-27 impairs the adipogenic lineage commitment via targeting lysyl oxidase.

The recruitment and commitment of mesenchymal stem cells and their terminal differentiation into adipocytes are the main pathways for increasing adipocyte cell numbers during obesity. Our previous studies have shown that lysyl oxidase (Lox) is upregulated and functions as an essential factor during bone morphogenetic protein 4 (BMP4) -induced C3H10T1/2 cell adipocytic lineage commitment. However, the mechanism of Lox regulation during adipogenic lineage commitment has remained largely unestablished. Samples of adipose tissue from humans with different BMI and C57BL/6 mice with a high-fat diet were used to compare microRNA-27 (miR-27) expression level associated with obesity. Taqman assays were used for miR-27 expression detection and Oil Red O staining for adipogenesis analysis. A negative correlation was identified between Lox expression level and miR-27 expression in both BMP4-treated C3H10T1/2 cells and human subcutaneous adipose tissues. A Lox 3' UTR luciferase reporter assay showed that miR-27 directly targeted Lox. Furthermore, overexpression of miR-27 impaired BMP4-induced upregulation of Lox and adipocytic commitment, which could be rescued by overexpression of mature Lox. Conversely, miR-27 inhibition by specific inhibitors increased Lox expression and adipocytic commitment. Taken together, these results suggest a novel role for miR-27 in repressing adipogenic lineage commitment by targeting Lox.

The role of microRNAs in cell fate determination of mesenchymal stem cells: balancing adipogenesis and osteogenesis.

Mesenchymal stem cells (MSCs) are multipotent stem cells capable of differentiating into adipocytes, osteoblasts, or chondrocytes. A mutually inhibitory relationship exists between osteogenic and adipogenic lineage commitment and differentiation. Such cell fate decision is regulated by several signaling pathways, including Wnt and bone morphogenetic protein (BMP). Accumulating evidence indicates that microRNAs (miRNAs) act as switches for MSCs to differentiate into either osteogenic or adipogenic lineage. Different miRNAs have been reported to regulate a master transcription factor for osteogenesis, such as Runx2, as well as molecules in the Wnt or BMP signaling pathway, and control the balance between osteoblast and adipocyte differentiation. Here, we discuss recent advancement of the cell fate decision of MSCs by miRNAs and their targets. [BMB Reports 2015; 48(6): 319-323]

Heparin affects human bone marrow stromal cell fate: Promoting osteogenic and reducing adipogenic differentiation and conversion

Heparins are broadly used for the prevention and treatment of thrombosis and embolism. Yet, osteoporosis is considered to be a severe side effect in up to one third of all patients on long-term treatment. However, the mechanisms underlying this clinical problem are only partially understood. To investigate if heparin affects differentiation of skeletal precursors, we examined the effects of heparin on the osteogenic and adipogenic lineage commitment and differentiation of primary human bone marrow stromal cells (hBMSCs). Due to the known inverse relationship between adipogenesis and osteogenesis and the capacity of pre-differentiated cells to convert into the respective other lineage, we also determined heparin effects on osteogenic conversion and adipogenic differentiation/conversion. Interestingly, heparin did not only significantly increase mRNA expression and enzyme activity of the osteogenic marker alkaline phosphatase (ALP), but it also promoted mineralization during osteogenic differentiation and conversion. Furthermore, the mRNA expression of the osteogenic marker bone morphogenic protein 4 (BMP4) was enhanced. In addition, heparin administration partly prevented adipogenic differentiation and conversion demonstrated by reduced lipid droplet formation along with a decreased expression of adipogenic markers. Moreover, luciferase reporter assays, inhibitor experiments and gene expression analyses revealed that heparin had putative permissive effects on osteogenic signaling via the BMP pathway and reduced the mRNA expression of the Wnt pathway inhibitors dickkopf 1 (DKK1) and sclerostin (SOST). Taken together, our data show a rather supportive than inhibitory effect of heparin on osteogenic hBMSC differentiation and conversion in vitro. Further studies will have to investigate the net effects of heparin administration on bone formation versus bone resorption in vivo to unravel the molecular mechanisms of heparin-associated osteoporosis and reconcile conflicting experimental data with clinical observations.

The osteogenic or adipogenic lineage commitment of human mesenchymal stem cells is determined by protein kinase C delta.

BackgroundMesenchymal stem cells (MSCs) have the potential to differentiate into specialized cell lineages such as osteoblasts and adipocytes in vitro. There exists a reciprocal relationship between osteogenic and adipogenic differentiation of MSCs that an osteogenic phenotype occurs at the expense of an adipogenic phenotype and vice versa, which in turn influence one another’s phenotype through negative feedback loops. Thus, it is important to understand what signaling molecules modulate the lineage commitment of MSCs. Protein kinase C (PKC) plays a central role in cellular signal transduction for mediating diverse biological functions, and dysregulation of PKC activity is involved in various metabolic diseases including cancer, diabetes, and heart disease. Although the role of individual PKC isoforms has been investigated in various fields, the potential role of PKC in bone metabolism is not completely understood. In this study, we investigated the potential role of PKCδ in osteogenic lineage commitment of human bone marrow-derived mesenchymal stem cells (hBMSCs).ResultsWe observed that expression and phosphorylation of PKCδ were increased during osteogenic differentiation of hBMSCs. Pharmacological inhibition and genetic ablation of PKCδ in hBMSCs resulted in a significant attenuation of osteogenic differentiation as evidenced by reduced ALP activity and ECM mineralization, as well as down-regulation of the expression of osteoblast-specific genes. These effects were also accompanied by induction of adipogenic differentiation and up-regulation of the expression of adipocyte-specific genes involved in lipid synthesis in osteogenic induction of hBMSCs. Additionally, the activation of AMPK, which is a key cellular energy sensor, induced osteogenesis of hBMSCs. However, the inhibition of AMPK activity by compound C did not affect the activation of PKCδ at all, indicating that there is no direct correlation between AMPK and PKCδ in osteogenesis of hBMSCs.ConclusionsThese results suggest that PKCδ is a critical regulator for the balance between osteogenesis and adipogenesis of hBMSCs and thus has a potential novel therapeutic target for the treatment of metabolic bone diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12860-014-0042-4) contains supplementary material, which is available to authorized users.

Oestrogen signalling in white adipose progenitor cells inhibits differentiation into brown adipose and smooth muscle cells.

Oestrogen, often via oestrogen receptor alpha (ERα) signalling, regulates metabolic physiology, highlighted by post-menopausal temperature dysregulation (hot flashes), glucose intolerance, increased appetite and reduced metabolic rate. Here we show that ERα signalling has a role in adipose lineage specification in mice. ERα regulates adipose progenitor identity and potency, promoting white adipogenic lineage commitment. White adipose progenitors lacking ERα reprogramme and enter into smooth muscle and brown adipogenic fates. Mechanistic studies highlight a TGFβ programme involved in progenitor reprogramming downstream of ERα signalling. The observed reprogramming has profound metabolic outcomes; both female and male adipose-lineage ERα-mutant mice are lean, have improved glucose sensitivity and are resistant to weight gain on a high-fat diet. Further, they are hypermetabolic, hyperphagic and hyperthermic, all consistent with a brown phenotype. Together, these findings indicate that ERα cell autonomously regulates adipose lineage commitment, brown fat and smooth muscle cell formation, and systemic metabolism, in a manner relevant to prevalent metabolic diseases.

Histone deacetylase inhibition destabilizes the multi-potent state of uncommitted adipose-derived mesenchymal stromal cells.

Human adipose-derived mesenchymal stromal cells (AMSCs) grown in platelet lysate are promising agents for therapeutic tissue regeneration. Here, we investigated whether manipulation of epigenetic events by the clinically relevant histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) alters differentiation of AMSCs. The multipotency of AMSCs was validated by their ability to differentiate into osteogenic, chondrogenic, and adipogenic lineages. High-throughput RNA sequencing and RT-qPCR established that human histone deacetylases (HDAC1 to HDAC11, and SIRT1 to SIRT7) are differentially expressed in AMSCs. SAHA induces hyper-acetylation of histone H3 and H4, stimulates protein expression of the HDAC-responsive gene SLC9A3R1/NHERF1 and modulates the AKT/FOXO1 pathway. Biologically, SAHA interferes with osteogenic, chondrogenic and adipogenic lineage commitment of multipotent AMSCs. Mechanistically, SAHA-induced loss of differentiation potential of uncommitted AMSCs correlates with multiple changes in the expression of principal transcription factors that control mesenchymal or pluripotent states. We propose that SAHA destabilizes the multi-potent epigenetic state of uncommitted human AMSCs by hyper-acetylation and perturbation of key transcription factor pathways. Furthermore, AMSCs grown in platelet lysate may provide a useful biological model for screening of new HDAC inhibitors that control the biological fate of human mesenchymal stromal cells.

Review of Signaling Pathways Governing MSC Osteogenic and Adipogenic Differentiation

Mesenchymal stem cells (MSC) are multipotent cells, functioning as precursors to a variety of cell types including adipocytes, osteoblasts, and chondrocytes. Between osteogenic and adipogenic lineage commitment and differentiation, a theoretical inverse relationship exists, such that differentiation towards an osteoblast phenotype occurs at the expense of an adipocytic phenotype. This balance is regulated by numerous, intersecting signaling pathways that converge on the regulation of two main transcription factors: peroxisome proliferator-activated receptor-γ (PPARγ) and Runt-related transcription factor 2 (Runx2). These two transcription factors, PPARγ and Runx2, are generally regarded as the master regulators of adipogenesis and osteogenesis. This review will summarize signaling pathways that govern MSC fate towards osteogenic or adipocytic differentiation. A number of signaling pathways follow the inverse balance between osteogenic and adipogenic differentiation and are generally proosteogenic/antiadipogenic stimuli. These include β-catenin dependent Wnt signaling, Hedgehog signaling, and NELL-1 signaling. However, other signaling pathways exhibit more context-dependent effects on adipogenic and osteogenic differentiation. These include bone morphogenic protein (BMP) signaling and insulin growth factor (IGF) signaling, which display both proosteogenic and proadipogenic effects. In summary, understanding those factors that govern osteogenic versus adipogenic MSC differentiation has significant implications in diverse areas of human health, from obesity to osteoporosis to regenerative medicine.

MiR-17-5p and miR-106a are involved in the balance between osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) can differentiate into several distinct cell types, including osteoblasts and adipocytes. The balance between osteogenic and adipogenic differentiation is disrupted in several osteogenic-related disorders, such as osteoporosis. So far, little is known about the molecular mechanisms that drive final lineage commitment of MSCs. In this study, we revealed that miR-17-5p and miR-106a have dual functions in the modulation of human adipose-derived mesenchymal stem cells (hADSCs) commitment by gain- and loss-of-function assays. They could promote adipogenesis and inhibit osteogenesis. Luciferase reporter assay, western blot and ELISA suggested BMP2 was a direct target of miR-17-5p and miR-106a. Downregulation of endogeneous BMP2 by RNA interference suppressed osteogenesis and increased adipogenesis, similar to the effect of miR-17-5p and miR-106a upregulation. Moreover, the inhibitory effects of miR-17-5p on osteogenic and adipogenic differentiation of hADSCs could be reversed by BMP2 RNA interference. In conclusion, miR-17-5p and miR-106a regulate osteogenic and adipogenic lineage commitment of hADSCs by directly targeting BMP2, and subsequently decreased osteogenic TAZ, MSX2 and Runx2, and increased adipogenic C/EBPα and PPARγ.

High-Content Imaging-Based Screening of Microenvironment-Induced Changes to Stem Cells

Effective screening methodologies for cells are challenged by the divergent and heterogeneous nature of phenotypes inherent to stem cell cultures, particularly on engineered biomaterial surfaces. In this study, we showcase a high-content, confocal imaging-based methodology to parse single-cell phenotypes by quantifying organizational signatures of specific subcellular reporter proteins and applied this profiling approach to three human stem cell types (embryonic–human embryonic stem cell [hESC], induced pluripotent–induced pluripotent stem cell [iPSC], and mesenchymal–human mesenchymal stem cell [hMSC]). We demonstrate that this method could distinguish self-renewing subpopulations of hESCs and iPSCs from heterogeneous populations. This technique can also provide insights into how incremental changes in biomaterial properties, both physiochemical and mechanical, influence stem cell fates by parsing the organization of stem cell proteins. For example, hMSCs cultured on polymeric films with varying degrees of poly(ethylene glycol) to modulate osteogenic differentiation were parsed using high-content organization of the cytoskeletal protein F-actin. In addition, hMSCs cultured on a self-assembled monolayer platform featuring compositional gradients were screened and descriptors obtained to correlate substrate variations with adipogenic lineage commitment. Taken together, high-content imaging of structurally sensitive proteins can be used as a tool to identify stem cell phenotypes at the single-cell level across a diverse range of culture conditions and microenvironments.

Glucose Reduction Prevents Replicative Senescence and Increases Mitochondrial Respiration in Human Mesenchymal Stem Cells

The unique self-renewal and multilineage differentiation potential of mesenchymal stem cells (MSCs) make them a promising candidate for cell therapy applications. However, during in vitro expansion of MSCs, replicative senescence may occur and will compromise the quality of the expanded cells. Because calorie restriction has been shown to effectively extend the life span of various organisms, the purpose of this study is to investigate the effect of glucose reduction on MSCs and the coordinated changes in energy utilization. It was found that the frequency of cycling cells was significantly increased, while senescence markers such as β-galactosidase activities and p16(INK4a) expression level were markedly reduced in MSCs under low-glucose culture condition. Quantitative real-time PCR analysis demonstrated the preserved trilineage differentiation potentials of MSCs after low-glucose treatment. Interestingly, the ability of osteogenic lineage commitment was improved, while the ability of adipogenic lineage commitment was delayed in MSCs after glucose reduction. In addition, we observed decreased lactate production, increased electron transport chain complexes expression, and increased oxygen consumption in MSCs after glucose reduction treatment. Increased antioxidant defensive responses were evidenced by increased antioxidant enzymes expression and decreased superoxide production after glucose reduction. Taken together, our findings suggest that MSCs utilize energy more efficiently under restricted glucose treatment and exhibit greater self-renewal and antisenescence abilities, while their differentiation potentials remain unaffected.

MicroRNAs in the Regulation of Adipogenesis and Obesity

Worldwide obesity is a growing health problem, associated with increased risk of chronic disease. Understanding the molecular basis of adipogenesis and fat cell development in obesity is essential to identify new biomarkers and therapeutic targets for the development of anti-obesity drugs. microRNAs (miRNAs) appear to play regulatory roles in many biological processes associated with obesity, including adipocyte differentiation, insulin action and fat metabolism. Recent studies show miRNAs are dysregulated in obese adipose tissue. During adipogenesis miRNAs can accelerate or inhibit adipocyte differentiation and hence regulate fat cell development. In addition miRNAs may regulate adipogenic lineage commitment in multipotent stem cells and hence govern fat cell numbers. Recent findings suggest miR-519d may be associated with human obesity, but larger case-control studies are needed. Few miRNA targets have been experimentally validated in adipocytes but interestingly both miR-27 and miR-519d target PPAR family members, which are well established regulators of fat cell development. In this review recent advances in our understanding of the role of miRNAs in fat cell development and obesity are discussed. The potential of miRNA based therapeutics targeting obesity is highlighted as well as recommendations for future research which could lead to a breakthrough in the treatment of obesity.

Origin of Thymic Adipocytes in Aging: Incidental to Thymopoiesis or Instigator of Immunosenescence? (132.22)

Abstract Vishwa Deep Dixit, Yun-Hee Youm, Hyunwon Yang, Christo Venkov, Nancy R Manley, Eric Nielson, Todd Leff, Bolormaa Vandanmagsar By 50 years of age most of human thymus is replaced with adipocytes of unknown provenance and uncertain function. We investigated the lineage and mechanism of thymic adipocyte formation during aging and asked whether manipulating these pathways regulates thymic function. Using.We demonstrate that indelibly LacZ marked FoxN1 thymic epithelial cells transition to give rise to local tissue fibroblasts via the epithelial-mesenchymal transition(EMT)process. Importantly, we found an age-related increase in pro-EMT regulator FSP1 and pro-adipogenic transcription factor PPARγ in thymus. Consistent with these findings, a subset of FoxN1LacZ+ cells are FSP1+ with large unilocular lipid droplet, typical of adipocyte phenotype. Furthermore, aged FSP1.GFP reporter mice revealed that the sorted FSP1+ thymic fibroblasts lack TEC signatures and express PPARγ, indicative of adipogenic lineage commitment. Notably, overexpression of constitutive-active PPARγ in FSP1+ adipogenic lineage cells led to increased thymic adipogenesis and reduced thymopoiesis. Conversely, inhibition of EMT and thymoadipogenesis via anti-aging intervention, caloric restriction prevented thymic aging and immunosenescence. Future longevity studies using specific genetic manipulation of these pathways may provide a definitive answer to the puzzle of age-related thymic adiposity and involution.