Adipogenic Precursors Research Articles

Connexin43 in mesenchymal lineage cells regulates body adiposity and energy metabolism in mice.

Connexin43 (Cx43) is the most abundant gap junction protein present in the mesenchymal lineage. In mature adipocytes, Cx43 mediates white adipose tissue (WAT) beiging in response to cold exposure and maintains the mitochondrial integrity of brown adipose tissue (BAT). We found that genetic deletion of Gja1 (Cx43 gene) in cells that give rise to chondro-osteogenic and adipogenic precursors driven by the Dermo1/Twist2 promoter led to lower body adiposity and partial protection against the weight gain and metabolic syndrome induced by a high-fat diet (HFD) in both sexes. These protective effects were related to increased locomotion, fuel utilization, energy expenditure, nonshivering thermogenesis, and better glucose tolerance in conditionally Gja1-ablated mice. Accordingly, Gja1-mutant mice exhibited reduced adipocyte hypertrophy, partially preserved insulin sensitivity, increased BAT lipolysis, and decreased whitening under HFD. This metabolic phenotype was not reproduced with more restricted Gja1 ablation in differentiated adipocytes, suggesting that Cx43 in adipocyte progenitors or other targeted cells restrains energy expenditures and promotes fat accumulation. These results reveal what we believe is a hitherto unknown action of Cx43 in adiposity, and offer a promising new pharmacologic target for improving metabolic balance in diabetes and obesity.

Optical diffraction tomography and Raman spectroscopy reveal distinct cellular phenotypes during white and brown adipocyte differentiation

White adipose tissue (WAT) and brown adipose tissue (BAT) are the primary types of fats in humans, and they play prominent roles in energy storage and thermogenesis, respectively. While the mechanisms of terminal adipogenesis are well understood, much remains unknown about the early stages of adipogenic differentiation. Label-free approaches, such as optical diffraction tomography (ODT) and Raman spectroscopy, offer the ability to retrieve morphological and molecular information at the single cell level without the negative effects of photobleaching and system perturbation due to introduction of fluorophores. In this study, we employed 3D ODT and Raman spectroscopy to gain deeper insights into the early stages of differentiation of human white preadipocytes (HWPs) and human brown preadipocytes (HBPs). We utilized ODT to retrieve morphological information, including cell dry mass and lipid mass, and Raman spectroscopy to obtain molecular information about lipids. Our findings reveal that HWPs and HBPs undergo dynamic and differential changes during the differentiation process. Notably, we found that HBPs accumulated lipids more rapidly and had a higher lipid mass than HWPs. Additionally, both cell types experienced an increase and subsequent decrease in cell dry mass during the first seven days, followed by an increase after day 7, which we attribute to the transformation of adipogenic precursors in the early stages. Finally, HBPs had higher lipid unsaturation levels than HWPs for the same differentiation timepoints. The insights gained from our study provide crucial contributions towards the advancement of new therapies for obesity and related diseases.

Transcriptional repression of the MRTF/SRF-cytoskeleton pathway by Rev-erba promotes beigeing via adipogenic progenitor fate switch

The circadian clock exerts temporal control in metabolic processes to maintain homeostasis, with its disruption leading to the development of obesity and insulin resistance. Adipose fibrosis in obese subjects exacerbates insulin resistance by restricting healthy tissue expansion and remodeling. Rev-erbα is a key circadian clock repressor that displays circadian rhythmic expression in adipose tissue, although its role in adipose tissue fibrosis is not known. Here we show that Rev-ebα inhibits the fibrogenic fate of adipocyte progenitors while promoting beige adipogenic potential via transcription repression of the MRTF/SRF-actin cytoskeleton axis. ChIP-seq analysis of Rev-erbα cistrome in adipogenic precursors identified its transcriptional control of pathways involved in cytoskeleton modulation, including established myofibroblast gene markers small muscle α-actin and Tropomysin. Gain-of-function of Rev-erbα in beige adipogenic precursors markedly suppressed MRTF/SRF activity with enhanced beige differentiation, while Rev-erbα silencing suppressed beige adipogenesis. Prrx1-Cre-mediated subcutaneous beige fat-selective Rev-erbα knock-in induced browning with augmented mitochondrial metabolism, resulting in increased energy expenditure, resistance to obesity and insulin sensitivity. Furthermore, single cell RNA-seq of beige progenitors with Rev-erbα overexpression revealed striking loss of preadipcyte-like (PAL) subpopulations with myofibroblastic characteristics that arise from pericyte lineage. PAL beige progenitors displayed enrichment of epithelial mesenchymal transition-related gene signature and contractile program as compared to preadipocytes. Preadipocyte population with Rev-erbα overexpression exhibited activated oxidative phosphorylation pathway with inhibition of myofibrolast characteristics. Taken together, our findings identify a novel regulatory function of Rev-erbα in modulating adipogenic progenitor fate choice that may promote adipose tissue beigeing while suppressing fibrotic complications. NIDDK grant 1R01DK112794, 1R01DK130499 and AR-DMRI 2023 T2D Innovation Grant This is the full abstract presented at the American Physiology Summit 2023 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.

Smad4-mediated angiogenesis facilitates the beiging of white adipose tissue in mice

Beige adipocytes are thermogenic with high expression of uncoupling protein 1 in the white adipose tissue (WAT), accompanied by angiogenesis. Previous studies showed that Smad4 is important for angiogenesis. Here we studied whether endothelial Smad4-mediated angiogenesis is involved in WAT beiging. Inducible knockout of endothelial cell (EC) selective Smad4 (Smad4 iEC-KO) was achieved by using the Smad4 Floxp/floxp and Tie2 CreERT2 mice. Beige fat induction achieved by cold or adrenergic agonist, and angiogenesis were attenuated in WAT of Smad4 iEC-KO mice, with the less proliferation of ECs and adipogenic precursors. RNA sequencing of human ECs showed that Smad4 is involved in angiogenesis-related pathways. Knockdown of SMAD4 attenuated the upregulation of VEGFA, PDGFA, and angiogenesis invitro. Treatment of human ECs with palmitic acid-induced Smad1/5 phosphorylation and the upregulation of core endothelial genes. Our study shows that endothelial Smad4 is involved in WAT beiging through angiogenesis and the expansion of adipose precursors into beige adipocytes.

Csf1 from marrow adipogenic precursors is required for osteoclast formation and hematopoiesis in bone.

Colony-stimulating factor 1 (Csf1) is an essential growth factor for osteoclast progenitors and an important regulator for bone resorption. It remains elusive which mesenchymal cells synthesize Csf1 to stimulate osteoclastogenesis. We recently identified a novel mesenchymal cell population, marrow adipogenic lineage precursors (MALPs), in bone. Compared to other mesenchymal subpopulations, MALPs expressed Csf1 at a much higher level and this expression was further increased during aging. To investigate its role, we constructed MALP-deficient Csf1 CKO mice using AdipoqCre. These mice had increased femoral trabecular bone mass, but their cortical bone appeared normal. In comparison, depletion of Csf1 in the entire mesenchymal lineage using Prrx1Cre led to a more striking high bone mass phenotype, suggesting that additional mesenchymal subpopulations secrete Csf1. TRAP staining revealed diminished osteoclasts in the femoral secondary spongiosa region of Csf1 CKOAdipoq mice, but not at the chondral-osseous junction nor at the endosteal surface of cortical bone. Moreover, Csf1 CKOAdipoq mice were resistant to LPS-induced calvarial osteolysis. Bone marrow cellularity, hematopoietic progenitors, and macrophages were also reduced in these mice. Taken together, our studies demonstrate that MALPs synthesize Csf1 to control bone remodeling and hematopoiesis.

308-OR: The Circadian Clock Exerts Coordinated Control of Beige Adipocyte Development via Cytoskeleton-MRTF/SRF Signaling Cascade

The circadian clock confers temporal regulation in metabolism, and its disruption leads to obesity and insulin resistance. In the current study, we identify that the opposing circadian clock regulators, transcription activator Bmal1 and its repressor Rev-erbα, exert concerted control of the actin cytoskeleton-MRTF/SRF pathway to drive beige adipocyte development, and that this clock regulatory axis is required for beige fat thermogenic capacity. Key components of the MRTF/SRF signaling display circadian oscillations in beige fat, and cistromic analyses revealed Bmal1 and Rev-erbα chromatin occupancy of genes involved in MRTF/SRF regulation. Genetic loss- or gain-of-functions of Bmal1 and Rev-erbα in adipogenic precursors markedly altered actin cytoskeleton organization. Bmal1 silencing inhibits F-actin formation and MRTF/SRF activity whereas its forced expression augments actin cytoskeleton. Notably, Bmal1 and Rev-erbα transcriptional control of the MRTF/SRF signaling modulates beige fat thermogenic capacity in vivo. Prrx1-Cre-mediated subcutaneous beige fat-selective Bmal1 ablation induced browning with augmented mitochondrial metabolism, resulting in resistance to obesity that improved insulin sensitivity. Conversely, overexpression of Bmal1 in beige depot suppressed the thermogenic program with adipose expansion and impaired glucose tolerance. In contrast, Rev-erbα gain-of-function phenocopied that of Bmal1 ablation, leading to browning of beige fat, resistance to obesity and insulin sensitivity. Mechanistically, we show that genetic loss of Bmal1 enhanced beige precursor differentiation and thermogenic induction, whereas Rev-erbα overexpression similarly promoted beige adipogenesis. Collectively, these findings uncover a novel concerted circadian clock control in beige adipocyte development that determines thermogenic capacity via the cytoskeleton-MRTF/SRF signaling cascade. Disclosure X.Xiong: None. W.Li: None. R.Liu: None. V.Yechoor: n/a. K.Ma: None. Funding NIH 1R01DK112794 to KM and DK097160 to VY

Identification of a regulatory pathway inhibiting adipogenesis via RSPO2

Healthy adipose tissue remodeling depends on the balance between de novo adipogenesis from adipogenic progenitor cells and the hypertrophy of adipocytes. De novo adipogenesis has been shown to promote healthy adipose tissue expansion, which confers protection from obesity-associated insulin resistance. Here, we define the role and trajectory of different adipogenic precursor subpopulations and further delineate the mechanism and cellular trajectory of adipogenesis, using single-cell RNA-sequencing datasets of murine adipogenic precursors. We identify Rspo2 as a functional regulator of adipogenesis, which is secreted by a subset of CD142+ cells to inhibit maturation of early progenitors through the receptor Lgr4. Increased circulating RSPO2 in mice leads to adipose tissue hypertrophy and insulin resistance and increased RSPO2 levels in male obese individuals correlate with impaired glucose homeostasis. Taken together, these findings identify a complex cellular crosstalk that inhibits adipogenesis and impairs adipose tissue homeostasis.

TNFα Mediates Inflammation-Induced Effects on PPARG Splicing in Adipose Tissue and Mesenchymal Precursor Cells.

Low-grade chronic inflammation and reduced differentiation capacity are hallmarks of hypertrophic adipose tissue (AT) and key contributors of insulin resistance. We identified PPARGΔ5 as a dominant-negative splicing isoform overexpressed in the AT of obese/diabetic patients able to impair adipocyte differentiation and PPARγ activity in hypertrophic adipocytes. Herein, we investigate the impact of macrophage-secreted pro-inflammatory factors on PPARG splicing, focusing on PPARGΔ5. We report that the epididymal AT of LPS-treated mice displays increased PpargΔ5/cPparg ratio and reduced expression of Pparg-regulated genes. Interestingly, pro-inflammatory factors secreted from murine and human pro-inflammatory macrophages enhance the PPARGΔ5/cPPARG ratio in exposed adipogenic precursors. TNFα is identified herein as factor able to alter PPARG splicing—increasing PPARGΔ5/cPPARG ratio—through PI3K/Akt signaling and SRp40 splicing factor. In line with in vitro data, TNFA expression is higher in the SAT of obese (vs. lean) patients and positively correlates with PPARGΔ5 levels. In conclusion, our results indicate that inflammatory factors secreted by metabolically-activated macrophages are potent stimuli that modulate the expression and splicing of PPARG. The resulting imbalance between canonical and dominant negative isoforms may crucially contribute to impair PPARγ activity in hypertrophic AT, exacerbating the defective adipogenic capacity of precursor cells.

Perspectives on scaling production of adipose tissue for food applications

With rising global demand for food proteins and significant environmental impact associated with conventional animal agriculture, it is important to develop sustainable alternatives to supplement existing meat production. Since fat is an important contributor to meat flavor, recapitulating this component in meat alternatives such as plant based and cell cultured meats is important. Here, we discuss the topic of cell cultured or tissue engineered fat, growing adipocytes in vitro that could imbue meat alternatives with the complex flavor and aromas of animal meat. We outline potential paths for the large scale production of in vitro cultured fat, including adipogenic precursors during cell proliferation, methods to adipogenically differentiate cells at scale, as well as strategies for converting differentiated adipocytes into 3D cultured fat tissues. We showcase the maturation of knowledge and technology behind cell sourcing and scaled proliferation, while also highlighting that adipogenic differentiation and 3D adipose tissue formation at scale need further research. We also provide some potential solutions for achieving adipose cell differentiation and tissue formation at scale based on contemporary research and the state of the field.

Smad2/3 Activation Regulates Smad1/5/8 Signaling via a Negative Feedback Loop to Inhibit 3T3-L1 Adipogenesis

Adipose tissues (AT) expand in response to energy surplus through adipocyte hypertrophy and hyperplasia. The latter, also known as adipogenesis, is a process by which multipotent precursors differentiate to form mature adipocytes. This process is directed by developmental cues that include members of the TGF-β family. Our goal here was to elucidate, using the 3T3-L1 adipogenesis model, how TGF-β family growth factors and inhibitors regulate adipocyte development. We show that ligands of the Activin and TGF-β families, several ligand traps, and the SMAD1/5/8 signaling inhibitor LDN-193189 profoundly suppressed 3T3-L1 adipogenesis. Strikingly, anti-adipogenic traps and ligands engaged the same mechanism of action involving the simultaneous activation of SMAD2/3 and inhibition of SMAD1/5/8 signaling. This effect was rescued by the SMAD2/3 signaling inhibitor SB-431542. By contrast, although LDN-193189 also suppressed SMAD1/5/8 signaling and adipogenesis, its effect could not be rescued by SB-431542. Collectively, these findings reveal the fundamental role of SMAD1/5/8 for 3T3-L1 adipogenesis, and potentially identify a negative feedback loop that links SMAD2/3 activation with SMAD1/5/8 inhibition in adipogenic precursors.

200-LB: Circadian Regulation of the MRTF-SRF Signaling Promotes Beige Adipocyte Development

The adipogenic progenitor transformation into mature adipocytes requires dissolution of intracellular actin cytoskeleton, and the cytoskeleton-MRTF/SRF signaling blocks adipogenesis. The circadian clock confers temporal control in metabolism, with tissue-intrinsic clock circuits contributing to metabolic homeostasis. In the current study, we identify a novel clock-MRTF/SRF regulatory axis that suppresses beige adipogenesis that is required for whole-body glucose metabolism. Key components of the cytoskeleton-MRTF/SRF signaling cascade display circadian oscillations in beige fat depot mediated by Bmal1 transcriptional activity. Genetic loss- or gain-of-functions of Bmal1 in adipogenic precursors markedly altered actin cytoskeleton organization, with silencing of Bmal1 inhibiting F-actin formation and MRTF/SRF activity whereas its forced expression augmenting actin architecture. Furthermore, we show that Bmal1 circadian control of the MRTF/SRF pathway drives beige thermogenic capacity in vivo. Prrx1-Cre-mediated beige fat-selective Bmal1 ablation induced beige depot browning with augmented mitochondrial metabolism, resulting in improved whole-body insulin sensitivity and resistance to obesity. Conversely, beige fat-specific overexpression of Bmal1 suppressed beige thermogenic program leading to impaired glucose tolerance and adipose expansion. Mechanistically, we show that Bmal1 deficiency enhanced beige precursor differentiation and thermogenic induction. Collectively, our findings uncover a temporal regulation of the cytoskeleton-MRTF/SRF signaling that modulates beige thermogenic capacity to maintain metabolic homeostasis. Disclosure X. Xiong: None. R. Liu: None. V. Yechoor: None. P. Saha: None. K. Ma: None. Funding National Institutes of Health (DK112794, DK097160-01); American Heart Association (17GRNT33370012)

Global transcriptomic analysis reveals Lnc-ADAMTS9 exerting an essential role in myogenesis through modulating the ERK signaling pathway

BackgroundLong non-coding RNAs (lncRNAs) are emerging key regulators involved in a variety of biological processes such as cell differentiation and development. The balance between myogenesis and adipogenesis is crucial for skeletal muscle homeostasis in humans and meat quality in farm animals. The present study aimed to reveal the global transcriptomic profiles of adipogenic (Adi-) and myogenic (Myo-) precursors derived from porcine skeletal muscle and identify lncRNAs involved in the modulation of myogenesis homeostasis in porcine skeletal muscle.ResultsIn this study, a total of 655 novel individual lncRNAs including differentially expressed 24 lncRNAs, and 755 differentially expressed mRNAs were identified (fold change ≥2 or ≤ 0.5 and adjusted P < 0.05). Integrated results of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis accompanied by the variation of intracellular Ca2+ concentration highlighted Lnc-ADAMTS9 involved in the modulation of myogenesis homeostasis in porcine skeletal muscle. Although Lnc-ADAMTS9 knock-down did not alter the mRNA expression of ADAMTS9, we demonstrated that Lnc-ADAMTS9 can promote myogenic proliferation and myogenic differentiation of myogenic precursors through inhibiting the ERK/MAPK signaling pathway.ConclusionWe deciphered a comprehensive catalog of mRNAs and lncRNAs that might be involved in the regulation of myogenesis and adipogenesis homeostasis in the skeletal muscle of pigs. The Lnc-ADAMTS9 exerts an essential role in myogenesis through the ERK signaling pathway.

Global DNA methylation pattern involved in the modulation of differentiation potential of adipogenic and myogenic precursors in skeletal muscle of pigs

BackgroundSkeletal muscle is a complex and heterogeneous tissue accounting for approximately 40% of body weight. Excessive ectopic lipid accumulation in the muscle fascicle would undermine the integrity of skeletal muscle in humans but endow muscle with marbling-related characteristics in farm animals. Therefore, the balance of myogenesis and adipogenesis is of great significance for skeletal muscle homeostasis. Significant DNA methylation occurs during myogenesis and adipogenesis; however, DNA methylation pattern of myogenic and adipogenic precursors derived from skeletal muscle remains unknown yet.MethodsIn this study, reduced representation bisulfite sequencing was performed to analyze genome-wide DNA methylation of adipogenic and myogenic precursors derived from the skeletal muscle of neonatal pigs. Integrated analysis of DNA methylation and transcription profiles was further conducted. Based on the results of pathway enrichment analysis, myogenic precursors were transfected with CACNA2D2-overexpression plasmids to explore the function of CACNA2D2 in myogenic differentiation.ResultsAs a result, 11,361 differentially methylated regions mainly located in intergenic region and introns were identified. Furthermore, 153 genes with different DNA methylation and gene expression level between adipogenic and myogenic precursors were characterized. Subsequently, pathway enrichment analysis revealed that DNA methylation programing was involved in the regulation of adipogenic and myogenic differentiation potential through mediating the crosstalk among pathways including focal adhesion, regulation of actin cytoskeleton, MAPK signaling pathway, and calcium signaling pathway. In particular, we characterized a new role of CACNA2D2 in inhibiting myogenic differentiation by suppressing JNK/MAPK signaling pathway.ConclusionsThis study depicted a comprehensive landmark of DNA methylome of skeletal muscle-derived myogenic and adipogenic precursors, highlighted the critical role of CACNA2D2 in regulating myogenic differentiation, and illustrated the possible regulatory ways of DNA methylation on cell fate commitment and skeletal muscle homeostasis.

Dynamic membrane proteome of adipogenic and myogenic precursors in skeletal muscle highlights EPHA2 may promote myogenic differentiation through ERK signaling

The balance of myogenic and adipogenic differentiation is crucial for skeletal muscle homeostasis. Given the vital role of membrane proteins (MBPs) in cell signal perception, membrane proteomics was conducted to delineate mechanisms regulating differentiation of adipogenic and myogenic precursors in skeletal muscle. Adipogenic and myogenic precursors with divergent differentiation potential were isolated from the longissimus dorsi muscle of neonatal pigs by the preplate method. A total of 85 differentially expressed MBPs (P < 0.05 and fold change ≥1.2 or ≤0.83) between 2 precursors were detected via isobaric tags for relative and absolute quantitation (iTRAQ) assay, including 67 up-regulated and 18 down-regulated in myogenic precursors. Functional enrichment analysis uncovered that myogenic and adipogenic precursors showed significant differences in cytoskeleton organization, syncytium formation, environmental information processing, and organismal systems. Furthermore, key MBPs in regulating cell differentiation were also characterized, including ITGB3, ITGAV, ITPR3, and EPHA2. Noteworthily, EPHA2 was required for myogenic differentiation, and it may promote myogenic differentiation through ERK signaling. Collectively, our study provided an insight into the distinct MBP profile between myogenic and adipogenic precursors in skeletal muscle and served as a solid basis for supporting the role of MBPs in regulating differentiation.—Zhang, X., Wang, L., Qiu, K., Xu, D., Yin, J. Dynamic membrane proteome of adipogenic and myogenic precursors in skeletal muscle highlights EPHA2 may promote myogenic differentiation through ERK signaling.

A Renewable Source of Human Beige Adipocytes for Development of Therapies to Treat Metabolic Syndrome

SUMMARYMolecular- and cellular-based therapies have the potential to reduce obesity-associated disease. In response to cold, beige adipocytes form in subcutaneous white adipose tissue and convert energy stored in metabolic substrates to heat, making them an attractive therapeutic target. We developed a robust method to generate a renewable source of human beige adipocytes from induced pluripotent stem cells (iPSCs). Developmentally, these cells are derived from FOXF1+ mesoderm and progress through an expandable mural-like mesenchymal stem cell (MSC) to form mature beige adipocytes that display a thermogenically active profile. This includes expression of uncoupling protein 1 (UCP1) concomitant with increased uncoupled respiration. With this method, dysfunctional adipogenic precursors can be reprogrammed and differentiated into beige adipocytes with increased thermogenic function and anti-diabetic secretion potential. This resource can be used to (1) elucidate mechanisms that underlie the control of beige adipogenesis and (2) generate material for cellular-based therapies that target metabolic syndrome in humans.

Protein Expression Landscape Defines the Differentiation Potential Specificity of Adipogenic and Myogenic Precursors in the Skeletal Muscle.

The balance of adipogenic and myogenic differentiation of skeletal muscle-derived mesenchymal stem cells is particularly important in muscle development and intramuscular fat deposition. This study aimed to explore the differential regulation between adipogenic and myogenic precursors by comparative analysis of their global proteome expression profile. Adipogenic and myogenic precursors isolated from neonatal porcine longissimus dorsi muscle by the preplate method were verified for their unique and distinct differentiation potential under myogenic or adipogenic induction. A total of 433 differentially expressed proteins (DEP) ( P < 0.05 and FC > 1.20 or <0.83) between adipogenic and myogenic precursors were detected via a tandem mass tag (TMT)-coupled LC-MS/MS approach, including 339 up-regulated and 94 down-regulated proteins in myogenic precursors compared with adipogenic precursors. On the basis of functional annotation and enrichment analysis of 433 DEP, adipogenic and myogenic precursors showed significantly different metabolic pattern of energy substances and differential regulations of gene expression, cell structure and development, ion homeostasis, and cell motility and migration. Three pathways including PPAR signaling pathway, phosphatidylinositol signaling system, and autophagy signaling pathway, which was differentially regulated between adipogenic and myogenic precursors, was also discovered to play crucial roles in cell differentiation. In conclusion, these differentiated regulation patterns between the two cell subsets of mesenchymal precursors together defines their differentiation potential specificity.

Automated microscopy as a quantitative method to measure differences in adipogenic differentiation in preparations of human mesenchymal stromal cells

Background aimsMultipotent stromal cells, also called mesenchymal stromal cells (MSCs), are potentially valuable as a cellular therapy because of their differentiation and immunosuppressive properties. As the result of extensive heterogeneity of MSCs, quantitative approaches to measure differentiation capacity between donors and passages on a per-cell basis are needed. MethodsHuman bone marrow-derived MSCs were expanded to passages P3, P5 and P7 from eight different donors and were analyzed for colony-forming unit capacity (CFU), cell size, surface marker expression and forward/side-scatter analysis by flow cytometry. Adipogenic differentiation potential was quantified with the use of automated microscopy. Percentage of adipogenesis was determined by quantifying nuclei and Nile red–positive adipocytes after automated image acquisition. ResultsMSCs varied in expansion capacity and increased in average cell diameter with passage. CFU capacity decreased with passage and varied among cell lines within the same passage. The number of adipogenic precursors varied between cell lines, ranging from 0.5% to 13.6% differentiation at P3. Adipogenic capacity decreased significantly with increasing passage. MSC cell surface marker analysis revealed no changes caused by passaging or donor differences. ConclusionsWe measured adipogenic differentiation on a per-cell basis with high precision and accuracy with the use of automated fluorescence microscopy. We correlated these findings with other quantitative bioassays to better understand the role of donor variability and passaging on CFU, cell size and adipogenic differentiation capacity in vitro. These quantitative approaches provide valuable tools to measure MSC quality and measure functional biological differences between donors and cell passages that are not revealed by conventional MSC cell surface marker analysis.

Proteomic techniques for characterisation of mesenchymal stem cell secretome

Mesenchymal stem cells (MSCs) are multipotent cells with a substantial potential in human regenerative medicine due to their ability to migrate to sites of injury, capability to suppress immune response and accessibility in large amount from patient's own bone marrow or fat tissue. It has been increasingly observed that the transplanted MSCs did not necessarily engraft and differentiate at the site of injury but might exert their therapeutic effects through secreted trophic signals. The MSCs secrete a variety of autocrine/paracrine factors, called secretome, that support regenerative processes in the damaged tissue, induce angiogenesis, protect cells from apoptotic cell death and modulate immune system. The cell culture medium conditioned by MSCs or osteogenic, chondrogenic as well as adipogenic precursors derived from MSCs has become a subject of intensive proteomic profiling in the search for and identification of released factors and microvesicles that might be applicable in regenerative medicine. Jointly with the methods for MSC isolation, expansion and differentiation, proteomic analysis of MSC secretome was enabled recently mainly due to the extensive development in protein separation techniques, mass spectrometry, immunological methods and bioinformatics. This review describes proteomic techniques currently applied or prospectively applicable in MSC secretomics, with a particular focus on preparation of the secretome sample, protein/peptide separation, mass spectrometry and protein quantification techniques, analysis of posttranslational modifications, immunological techniques, isolation and characterisation of secreted vesicles and exosomes, analysis of cytokine-encoding mRNAs and bioinformatics.

Modulation of Adipogenic Conditions for Prospective Use of hADSCs in Adipose Tissue Engineering

Modern strategies in adipose tissue engineering (ATE) take advantage of the easy harvest, abundance and differentiation potential towards mesenchymal lineages of hADSCs. The controlled conversion of hADSCs to committed adipogenic precursors and further mature adipocytes formation is important for good long-term results in soft tissue regeneration. Thus, in this study, we report: (i) the isolation of the processed lipoaspirate (PLA) cells from adipose tissue and sanguine fractions; (ii) the phenotypic characterization of the PLA descendants; (iii) the design of a novel protocol for the modulation of adipogenic conditions in the perspectives of ATE applications. To modulate the differentiation rate through our protocol, we propose to selectively modify the formulation of the adipogenic media in accordance with the evolution of the process. Therefore, we aimed to ensure the long-term proliferation of the precursor cells and to delay the late adipogenic events. The status of differentiation was characterized in terms of intracellular lipid accumulation and reorganization of the cytoskeleton simultaneously with perilipin protein expression. Moreover, we studied the sequential activation of PPARγ2, FAS, aP2 and perilipin genes which influence the kinetics of the adipogenic process. The strategies developed in this work are the prerequisites for prospective 3D regenerative systems.

Horse serum reduces expression of membrane-bound and soluble isoforms of the preadipocyte marker Delta-like 1 homolog (Dlk1), but is inefficient for adipogenic differentiation of mouse preadipocytes

Downregulation of the preadipocyte marker Delta-like 1 homologue (Dlk1), an inhibitor of adipogenesis, has been suggested to be a prerequisite for adipogenic differentiation to occur, and low Dlk1 levels are often used to verify adipogenesis. Mouse preadipocytic cell lines such as 3T3-L1, as well as primary derived preadipocytes, are important models to study adipogenic differentiation and obesity. However, in vitro adipogenic differentiation of primary derived preadipocytes remains incomplete, and identification of factors that will improve the adipogenic differentiation process is thus of high value. In this study we show that horse serum fails to improve adipogenic differentiation of mouse preadipocytes (both 3T3-L1 cells and primary derived mouse preadipocytes) as otherwise reported for bone marrow derived adipogenic precursors. Unexpectedly, while Dlk1 levels were indeed decreased using horse serum, this did not correlate with a high degree of adipogenic differentiation. In conclusion, our novel results thus reveal that horse serum clearly is insufficient for adipogenic differentiation of mouse preadipocytes and that low levels of Dlk1 alone are a poor marker of mouse in vitro adipogenesis. We would also like to emphasize that it is very important for the field of cellular differentiation that researchers thoroughly investigate the effect of individual reagents in their protocols. Such data will increase understanding of the limitations and possibilities of individual systems.