Alternative fat: redefining adipocytes for biomanufacturing cultivated meat.

Cellular agriculture provides a potentially sustainable way of producing cultivated meat as an alternative protein source. In addition to muscle and connective tissue, fat is an important component of animal meat that contributes to taste, texture, tenderness, and nutritional profiles. However, while the biology of fat cells (adipocytes) is well studied, there is a lack of investigation on how adipocytes from agricultural species are isolated, produced, and incorporated as food constituents. Recently we compiled all protocols related to generation and analysis of adipose progenitors from bovine, porcine, chicken, other livestock and seafood species. In this review we summarize recent developments and present key scientific questions and challenges that need to be addressed in order to advance the biomanufacture of 'alternative fat'.

Reassessment of adipocyte technology for cellular agriculture of alternative fat.

Alternative proteins, such as cultivated meat, have recently attracted significant attention as novel and sustainable food. Fat tissue/cell is an important component of meat that makes organoleptic and nutritional contributions. Although adipocyte biology is relatively well investigated, there is limited focus on the specific techniques and strategies to produce cultivated fat from agricultural animals. In the assumed standard workflow, stem/progenitor cell lines are derived from tissues of animals, cultured for expansion, and differentiated into mature adipocytes. Here, we compile information from literature related to cell isolation, growth, differentiation, and analysis from bovine, porcine, chicken, other livestock, and seafood species. A diverse range of tissue sources, cell isolation methods, cell types, growth media, differentiation cocktails, and analytical methods for measuring adipogenic levels were used across species. Based on our analysis, we identify opportunities and challenges in advancing new technology era toward producing "alternative fat" that is suitable for human consumption.

Adipose Tissue: Understanding the Heterogeneity of Stem Cells for Regenerative Medicine.

Adipose-derived stem cells (ASCs) have been increasingly used as a versatile source of mesenchymal stem cells (MSCs) for diverse clinical investigations. However, their applications often become complicated due to heterogeneity arising from various factors. Cellular heterogeneity can occur due to: (i) nomenclature and criteria for definition; (ii) adipose tissue depots (e.g., subcutaneous fat, visceral fat) from which ASCs are isolated; (iii) donor and inter-subject variation (age, body mass index, gender, and disease state); (iv) species difference; and (v) study design (in vivo versus in vitro) and tools used (e.g., antibody isolation and culture conditions). There are also actual differences in resident cell types that exhibit ASC/MSC characteristics. Multilineage-differentiating stress-enduring (Muse) cells and dedifferentiated fat (DFAT) cells have been reported as an alternative or derivative source of ASCs for application in regenerative medicine. In this review, we discuss these factors that contribute to the heterogeneity of human ASCs in detail, and what should be taken into consideration for overcoming challenges associated with such heterogeneity in the clinical use of ASCs. Attempts to understand, define, and standardize cellular heterogeneity are important in supporting therapeutic strategies and regulatory considerations for the use of ASCs.

Novel live cell fluorescent probe for human-induced pluripotent stem cells highlights early reprogramming population.

BACKGROUND: Despite recent rapid progress in method development and biological understanding of induced pluripotent stem (iPS) cells, there has been a relative shortage of tools that monitor the early reprogramming process into human iPS cells. METHODS: We screened the in-house built fluorescent library compounds that specifically bind human iPS cells. After tertiary screening, the selected probe was analyzed for its ability to detect reprogramming cells in the time-dependent manner using high-content imaging analysis. The probe was compared with conventional dyes in different reprogramming methods, cell types, and cell culture conditions. Cell sorting was performed with the fluorescent probe to analyze the early reprogramming cells for their pluripotent characteristics and genome-wide gene expression signatures by RNA-seq. Finally, the candidate reprogramming factor identified was investigated for its ability to modulate reprogramming efficiency. RESULTS: We identified a novel BODIPY-derived fluorescent probe, BDL-E5, which detects live human iPS cells at the early reprogramming stage. BDL-E5 can recognize authentic reprogramming cells around 7 days before iPS colonies are formed and stained positive with conventional pluripotent markers. Cell sorting of reprogrammed cells with BDL-E5 allowed generation of an increased number and higher quality of iPS cells. RNA sequencing analysis of BDL-E5-positive versus negative cells revealed early reprogramming patterns of gene expression, which notably included CREB1. Reprogramming efficiency was significantly increased by overexpression of CREB1 and decreased by knockdown of CREB1. CONCLUSION: Collectively, BDL-E5 offers a valuable tool for delineating the early reprogramming pathway and clinically applicable commercial production of human iPS cells.

CD10 marks non-canonical PPARγ-independent adipocyte maturation and browning potential of adipose-derived stem cells.

BACKGROUND: Effective stem cell therapy is dependent on the stem cell quality that is determined by their differentiation potential, impairment of which leads to poor engraftment and survival into the target cells. However, limitations in our understanding and the lack of reliable markers that can predict their maturation efficacies have hindered the development of stem cells as an effective therapeutic strategy. Our previous study identified CD10, a pro-adipogenic, depot-specific prospective cell surface marker of human adipose-derived stem cells (ASCs). Here, we aim to determine if CD10 can be used as a prospective marker to predict mature adipocyte quality and play a direct role in adipocyte maturation. METHODS: We first generated 14 primary human subject-derived ASCs and stable immortalized CD10 knockdown and overexpression lines for 4 subjects by the lentiviral transduction system. To evaluate the role of CD10 in adipogenesis, the adipogenic potential of the human subject samples were scored against their respective CD10 transcript levels. Assessment of UCP1 expression levels was performed to correlate CD10 levels to the browning potential of mature ASCs. Quantitative polymerase chain reaction (qPCR) and Western blot analysis were performed to determine CD10-dependent regulation of various targets. Seahorse analysis of oxidative metabolism and lipolysis assay were studied. Lastly, as a proof-of-concept study, we used CD10 as a prospective marker for screening nuclear receptor ligands library. RESULTS: We identified intrinsic CD10 levels as a positive determinant of adipocyte maturation as well as browning potential of ASCs. Interestingly, CD10 regulates ASC's adipogenic maturation non-canonically by modulating endogenous lipolysis without affecting the classical peroxisome proliferator-activated receptor gamma (PPARγ)-dependent adipogenic pathways. Furthermore, our CD10-mediated screening analysis identified dexamethasone and retinoic acid as stimulator and inhibitor of adipogenesis, respectively, indicating CD10 as a useful biomarker for pro-adipogenic drug screening. CONCLUSION: Overall, we establish CD10 as a functionally relevant ASC biomarker, which may be a prerequisite to identify high-quality cell populations for improving metabolic diseases.

Diversification of reprogramming trajectories revealed by parallel single-cell transcriptome and chromatin accessibility sequencing.

Cellular reprogramming suffers from low efficiency especially for the human cells. To deconstruct the heterogeneity and unravel the mechanisms for successful reprogramming, we adopted single-cell RNA sequencing (scRNA-Seq) and single-cell assay for transposase-accessible chromatin (scATAC-Seq) to profile reprogramming cells across various time points. Our analysis revealed that reprogramming cells proceed in an asynchronous trajectory and diversify into heterogeneous subpopulations. We identified fluorescent probes and surface markers to enrich for the early reprogrammed human cells. Furthermore, combinatory usage of the surface markers enabled the fine segregation of the early-intermediate cells with diverse reprogramming propensities. scATAC-Seq analysis further uncovered the genomic partitions and transcription factors responsible for the regulatory phasing of reprogramming process. Binary choice between a FOSL1 and a TEAD4-centric regulatory network determines the outcome of a successful reprogramming. Together, our study illuminates the multitude of diverse routes transversed by individual reprogramming cells and presents an integrative roadmap for identifying the mechanistic part list of the reprogramming machinery.

Trxlp, a thioredoxin-like effector from Edwardsiella piscicida inhibits cellular redox signaling and nuclear translocation of NF-κB.

Redox signaling and homeostasis are essential for cell survival and the immune response. Peroxiredoxin (Prx) modulates the level of H2O2 as a redox signal through H2O2 decomposition. The redox activity of thioredoxin (Trx) is required as a reducing equivalent to regenerate Prx. Edwardsiella piscicida is an opportunistic Gram-negative enteric pathogen that secretes a novel Trx-like effector protein, ETAE_2186 (Trxlp). Trxlp has unique structural properties compared with other Trx proteins. In enzymatic and binding assays, we confirmed Trxlp to be redox-inactive due to the low reactivity and flexibility of the resolving cysteine residue, C35, at the active site motif "31WCXXC35". We identified key residues near the active site that are critical for reactivity and flexibility of C35 by site-directed mutagenesis analysis. NMR titration experiment demonstrated prolong inhibitory interaction of Trxlp with Prx1 resulting in the repression of Prx1-mediated H2O2 decomposition leading to increased ROS accumulation in infected host cells. Increased ROS in turn prevented nuclear translocation of NF-κB and inhibition of NF-κB target genes, leading to bacterial survival and enhanced replication inside host cells. Targeting Trxlp-mediated virulence promises to attenuate E. piscicida infection.

Oxidative stress mediates depot-specific functional differences of human adipose-derived stem cells.

BACKGROUND: Visceral (VS) fat depot is known to have defective adipogenic functions compared to subcutaneous (SC) fat, but its mechanism of origin is unclear. OBJECTIVE: We tested our hypothesis that the degree of oxidative stress in adipose-derived stem cells (ASCs) from these depots may account for this difference. METHODS: ASCs were isolated from VS (omental region) and SC (abdominal region) fat depots of human subjects undergoing bariatric surgery. ASCs from VS and SC fat were investigated for their cellular characteristics in reactive oxygen species (ROS), metabolism, gene expression, proliferation, senescence, migration, and adipocyte differentiation. ASCs were also treated with antioxidant ascorbic acid (vitamin C). RESULTS: We found that human VS-derived ASCs exhibit excessive oxidative stress characterized by high reactive oxygen species (ROS), compared to SC-derived ASCs. Gene expression analyses indicate that the VS-ASCs exhibit higher levels of genes involved in pro-oxidant and pro-inflammatory pathways and lower levels of genes in antioxidant and anti-inflammatory pathways. VS-ASCs have impaired cellular functions compared to SC-ASCs, such as slower proliferation, early senescence, less migratory activity, and poor adipogenic capability in vitro. Treatment with ascorbic acid decreased ROS levels drastically in VS-ASCs. Ascorbic acid treatment substantially improved proliferation, senescence, migration, and adipogenic capacities of compromised ASCs caused by high ROS. CONCLUSIONS: This finding suggests the fat depot-specific differences of cellular defects originating from stem cell population. Considering clinical potentials of human ASCs for cell therapies, this also offers a possible strategy for improving their therapeutic qualities through antioxidants.

Fast Adipogenesis Tracking System (FATS)-a robust, high-throughput, automation-ready adipogenesis quantification technique.

Adipogenesis is essential in in vitro experimentation to assess differentiation capability of stem cells, and therefore, its accurate measurement is important. Quantitative analysis of adipogenic levels, however, is challenging and often susceptible to errors due to non-specific reading or manual estimation by observers. To this end, we developed a novel adipocyte quantification algorithm, named Fast Adipogenesis Tracking System (FATS), based on computer vision libraries. The FATS algorithm is versatile and capable of accurately detecting and quantifying percentage of cells undergoing adipogenic and browning differentiation even under difficult conditions such as the presence of large cell clumps or high cell densities. The algorithm was tested on various cell lines including 3T3-L1 cells, adipose-derived mesenchymal stem cells (ASCs), and induced pluripotent stem cell (iPSC)-derived cells. The FATS algorithm is particularly useful for adipogenic measurement of embryoid bodies derived from pluripotent stem cells and was capable of accurately distinguishing adipogenic cells from false-positive stains. We then demonstrate the effectiveness of the FATS algorithm for screening of nuclear receptor ligands that affect adipogenesis in the high-throughput manner. Together, the FATS offer a universal and automated image-based method to quantify adipocyte differentiation of different cell lines in both standard and high-throughput workflows.

Thyroid hormone (T3) stimulates brown adipose tissue activation via mitochondrial biogenesis and MTOR-mediated mitophagy.

The thyroid hormone triiodothyronine (T3) activates thermogenesis by uncoupling electron transport from ATP synthesis in brown adipose tissue (BAT) mitochondria. Although T3 can induce thermogenesis by sympathetic innervation, little is known about its cell autonomous effects on BAT mitochondria. We thus examined effects of T3 on mitochondrial activity, autophagy, and metabolism in primary brown adipocytes and BAT and found that T3 increased fatty acid oxidation and mitochondrial respiration as well as autophagic flux, mitophagy, and mitochondrial biogenesis. Interestingly, there was no significant induction of intracellular reactive oxygen species (ROS) despite high mitochondrial respiration and UCP1 induction by T3. However, when cells were treated with Atg5 siRNA to block autophagy, induction of mitochondrial respiration by T3 decreased, and was accompanied by ROS accumulation, demonstrating a critical role for autophagic mitochondrial turnover. We next generated an Atg5 conditional knockout mouse model (Atg5 cKO) by injecting Ucp1 promoter-driven Cre-expressing adenovirus into Atg5Flox/Flox mice to examine effects of BAT-specific autophagy on thermogenesis in vivo. Hyperthyroid Atg5 cKO mice exhibited lower body temperature than hyperthyroid or euthyroid control mice. Metabolomic analysis showed that T3 increased short and long chain acylcarnitines in BAT, consistent with increased ß-oxidation. T3 also decreased amino acid levels, and in conjunction with SIRT1 activation, decreased MTOR activity to stimulate autophagy. In summary, T3 has direct effects on mitochondrial autophagy, activity, and turnover in BAT that are essential for thermogenesis. Stimulation of BAT activity by thyroid hormone or its analogs may represent a potential therapeutic strategy for obesity and metabolic diseases. Abbreviations: ACACA: acetyl-Coenzyme A carboxylase alpha; AMPK: AMP-activated protein kinase; Acsl1: acyl-CoA synthetase long-chain family member 1; ATG5: autophagy related 5; ATG7: autophagy related 7; ATP: adenosine triphosphate; BAT: brown adipose tissue; cKO: conditional knockout; COX4I1: cytochrome c oxidase subunit 4I1; Cpt1b: carnitine palmitoyltransferase 1b, muscle; CQ: chloroquine; DAPI: 4',6-diamidino-2-phenylindole; DIO2: deiodinase, iodothyronine, type 2; DMEM: Dulbecco's modified Eagle's medium; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; Fabp4: fatty acid binding protein 4, adipocyte; FBS: fetal bovine serum; FCCP: carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; FGF: fibroblast growth factor; FOXO1: forkhead box O1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; Gpx1: glutathione peroxidase 1; Lipe: lipase, hormone sensitive; MAP1LC3B: microtubule-associated protein 1 light chain 3; mRNA: messenger RNA; MTORC1: mechanistic target of rapamycin kinase complex 1; NAD: nicotinamide adenine dinucleotide; Nrf1: nuclear respiratory factor 1; OCR: oxygen consumption rate; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PPARGC1A: peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; Pnpla2: patatin-like phospholipase domain containing 2; Prdm16: PR domain containing 16; PRKA: protein kinase, AMP-activated; RPS6KB: ribosomal protein S6 kinase; RFP: red fluorescent protein; ROS: reactive oxygen species; SD: standard deviation; SEM: standard error of the mean; siRNA: small interfering RNA; SIRT1: sirtuin 1; Sod1: superoxide dismutase 1, soluble; Sod2: superoxide dismutase 2, mitochondrial; SQSTM1: sequestosome 1; T3: 3,5,3'-triiodothyronine; TFEB: transcription factor EB; TOMM20: translocase of outer mitochondrial membrane 20; UCP1: uncoupling protein 1 (mitochondrial, proton carrier); ULK1: unc-51 like kinase 1; VDAC1: voltage-dependent anion channel 1; WAT: white adipose tissue.

A roundtable on responsible innovation with autologous stem cells in Australia, Japan and Singapore.

We report on a roundtable event hosted in Singapore that sought to identify some of the ethical and regulatory challenges in translating autologous cell-based interventions, particularly those claiming to involve stem cells, into safe and effective therapies and to propose some solutions to encourage responsible innovation with these products. Challenges are identified in the three areas of cell manufacturing and processing, innovative uses of autologous cells in clinical practice and standards of evidence. Proposed solutions are discussed within a co-operative model of statutory laws and regulations that can enable product development with autologous cells and professional codes and standards that can encourage ethical conduct in clinical practice. Future research should be directed toward establishing regional networks for the development of internationally consistent standards in manufacturing and ethical codes of conduct for innovating with stem cells, and other autologous cells, and fostering ongoing exchange between jurisdictions.

Quantitative in vivo detection of adipose tissue browning using diffuse reflectance spectroscopy in near-infrared II window.

White adipose tissue (WAT) and brown adipose tissue (BAT) biologically function in an opposite way in energy metabolism. BAT induces energy consumption by heat production while WAT mainly stores energy in the form of triglycerides. Recent progress in the conversion of WAT cells to "beige" or "brown-like" adipocytes in animals, having functional similarity to BAT, spurred a great interest in developing the next-generation therapeutics in the field of metabolic disorders. Though magnetic resonance imaging and positron emission tomography could detect classical BAT and WAT in animals and humans, it is of a great challenge in detecting the "browning" process in vivo. Here, to the best of our knowledge, for the first time, we present a simple, cost-effective, label-free fiber optic-based diffuse reflectance spectroscopy measurement in the near infrared II window (~1050-1400 nm) for the quantitative detection of browning in a mouse model in vivo. We could successfully quantify the browning of WAT in a mouse model by estimating the lipid fraction, which serves as an endogenous marker. Lipid fraction exhibited a gradual decrease from WAT to BAT with beige exhibiting an intermediate value. in vivo browning process was also confirmed with standard molecular and biochemical assays.

Multimodal imaging approach to monitor browning of adipose tissue in vivo.

The discovery that white adipocytes can undergo a browning process to become metabolically active beige cells has attracted significant interest in the fight against obesity. However, the study of adipose browning has been impeded by a lack of imaging tools that allow longitudinal and noninvasive monitoring of this process in vivo. Here, we report a preclinical imaging approach to detect development of beige adipocytes during adrenergic stimulation. In this approach, we expressed near-infrared fluorescent protein, iRFP720, driven under an uncoupling protein-1 (Ucp1) promoter in mice by viral transduction, and used multispectral optoacoustic imaging technology with ultrasound tomography (MSOT-US) to assess adipose beiging during adrenergic stimulation. We observed increased photoacoustic signal at 720 nm, coupled with attenuated lipid signals in stimulated animals. As a proof of concept, we validated our approach against hybrid positron emission tomography combined with magnetic resonance (PET/MR) imaging modality, and quantified the extent of adipose browning by MRI-guided segmentation of 2-deoxy-2-18F-fluoro-d-glucose uptake signals. The browning extent detected by MSOT-US and PET/MR are well correlated with Ucp1 induction. Taken together, these systems offer great opportunities for preclinical screening aimed at identifying compounds that promote adipose browning and translation of these discoveries into clinical studies of humans.

Amenable epigenetic traits of dental pulp stem cells underlie high capability of xeno-free episomal reprogramming.

BACKGROUND: While a shift towards non-viral and animal component-free methods of generating induced pluripotent stem (iPS) cells is preferred for safer clinical applications, there is still a shortage of reliable cell sources and protocols for efficient reprogramming. METHODS: Here, we show a robust episomal and xeno-free reprogramming strategy for human iPS generation from dental pulp stem cells (DPSCs) which renders good efficiency (0.19%) over a short time frame (13-18 days). RESULTS: The robustness of DPSCs as starting cells for iPS induction is found due to their exceptional inherent stemness properties, developmental origin from neural crest cells, specification for tissue commitment, and differentiation capability. To investigate the epigenetic basis for the high reprogramming efficiency of DPSCs, we performed genome-wide DNA methylation analysis and found that the epigenetic signature of DPSCs associated with pluripotent, developmental, and ecto-mesenchymal genes is relatively close to that of iPS and embryonic stem (ES) cells. Among these genes, it is found that overexpression of PAX9 and knockdown of HERV-FRD improved the efficiencies of iPS generation. CONCLUSION: In conclusion, our study provides underlying epigenetic mechanisms that establish a robust platform for efficient generation of iPS cells from DPSCs, facilitating industrial and clinical use of iPS technology for therapeutic needs.

miR-524-5p of the primate-specific C19MC miRNA cluster targets TP53IPN1- and EMT-associated genes to regulate cellular reprogramming.

BACKGROUND: Introduction of the transcription factors Oct4, Sox2, Klf4, and c-Myc (OSKM) is able to 'reprogram' somatic cells to become induced pluripotent stem cells (iPSCs). Several microRNAs (miRNAs) are known to enhance reprogramming efficiency when co-expressed with the OSKM factors. The primate-specific chromosome 19 miRNA cluster (C19MC) is essential in primate reproduction, development, and differentiation. miR-524-5p, a C19MC member, is highly homologous to the reprogramming miR-520d-5p; we also reported that miR-524-5p was expressed in iPSCs but not mesenchymal stem cells (MSCs). This study aimed to elucidate possible contributions of miR-524-5p to the reprogramming process. METHODS: A miR-524-5p precursor was introduced into human fibroblast HFF-1 in the presence of OSKM, and the relative number of embryonic stem cell (ESC)-like colonies that stained positively with alkaline phosphatase (AP) and Nanog were quantified to determine reprogramming efficiency. A miR-524-5p mimic was transfected to MSCs to investigate the effects of miR-524-5p on TP53INP1, ZEB2, and SMAD4 expression by real-time polymerase chain reaction (PCR) and Western blot. Direct gene targeting was confirmed by luciferase activity. A phylogenetic tree of TP53INP1 was constructed by the Clustal method. Contribution of miR-524-5p to cell proliferation and apoptosis was examined by cell counts, BrdU, MTT, and cell death assays, and pluripotency gene expression by real-time PCR. RESULTS: Co-expressing the miR-524 precursor with OSKM resulted in a two-fold significant increase in the number of AP- and Nanog-positive ESC-like colonies, indicating a role for miR-524-5p in reprogramming. The putative target, TP53INP1, showed an inverse expression relationship with miR-524-5p; direct TP53INP1 targeting was confirmed in luciferase assays. miR-524-5p-induced TP53INP1 downregulation enhanced cell proliferation, suppressed apoptosis, and upregulated the expression of pluripotency genes, all of which are critical early events of the reprogramming process. Interestingly, the TP53INP1 gene may have co-evolved late with the primate-specific miR-524-5p. miR-524-5p also promoted mesenchymal-to-epithelial transition (MET), a required initial event of reprogramming, by directly targeting the epithelial-to-mesenchymal transition (EMT)-related genes, ZEB2 and SMAD4. CONCLUSIONS: Via targeting TP53INP1, ZEB2, and SMAD4, miR-524-5p contributes to the early stage of inducing pluripotency by promoting cell proliferation, inhibiting apoptosis, upregulating expression of pluripotency genes, and enhancing MET. Other C19MC miRNAs may have similar reprogramming functions.

Selective activation of miRNAs of the primate-specific chromosome 19 miRNA cluster (C19MC) in cancer and stem cells and possible contribution to regulation of apoptosis.

BACKGROUND: The human chromosome 19 miRNA cluster (C19MC) of 43 genes is a primate-specific miRNA cluster that may have biological significance in the genetic complexity of the primate. Despite previous reports on individual C19MC miRNA expression in cancer and stem cells, systematic studies on C19MC miRNA expression and biological functions are lacking. RESULTS: Cluster-wide C19MC miRNA expression profiling by microarray analysis showed wholesome C19MC activation in embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). However, in multipotent adipose-derived mesenchymal stem cells (MSCs) and a unipotent human white pre-adipocyte cell line, only selected C19MC miRNAs were expressed. MiRNA copy number analysis also showed selective C19MC expression in cancer cells with expression patterns highly similar to those in MSCs, suggesting similar miRNA regulatory mechanisms in these cells. Selective miRNA expression also suggests complex transcriptional mechanism(s) regulating C19MC expression under specific cellular and pathological conditions. Bioinformatics analysis showed that sixteen of the C19MC miRNAs share the same "AAGUGC" seed sequence with members of the miR-302/-372 family, which are known cellular reprogramming factors. In particular, C19MC-AAGUGC-miRNAs with the nucleotides 2-7 canonical seed position as in miR-302/-372 miRNAs, may play similar roles as miR-302/-372 in induced pluripotency. A biased 3p-arm selection of the C19MC-AAGUGC-miRNAs was observed indicating that targets of the 3p species of these miRNAs may be biologically significant in regulating stemness. Furthermore, bioinformatics analysis of the putative targets of the C19MC-AAGUGC-miRNAs predicted significant involvement of signaling pathways in reprogramming, many of which contribute to promoting apoptosis by indirect activation of the pro-apoptotic proteins BAK/BAX via suppression of genes of the cell survival pathways, or by enhancing caspase-8 activation through targeting inhibitors of TRAIL-inducing apoptosis. CONCLUSIONS: This work demonstrated selective C19MC expression in MSCs and cancer cells, and, through miRNA profiling and bioinformatics analysis, predicted C19MC modulation of apoptosis in induced pluripotency and tumorigenesis.

Diffuse Optical Spectroscopy and Imaging to Detect and Quantify Adipose Tissue Browning.

Adipose (fat) tissue is a complex metabolic organ that is highly active and essential. In contrast to white adipose tissue (WAT), brown adipose tissue (BAT) is deemed metabolically beneficial because of its ability to burn calories through heat production. The conversion of WAT-resident adipocytes to "beige" or "brown-like" adipocytes has recently attracted attention. However, it typically takes a few days to analyze and confirm this browning of WAT through conventional molecular, biochemical, or histological methods. Moreover, accurate quantification of the overall browning process is not possible by any of these methods. In this context, we report the novel application of diffuse reflectance spectroscopy (DRS) and multispectral imaging (MSI) to detect and quantify the browning process in mice. We successfully demonstrated the time-dependent increase in browning of WAT, following its induction through ß-adrenergic agonist injections. The results from these optical techniques were confirmed with those of standard molecular and biochemical assays, which measure gene and protein expression levels of UCP1 and PGC-1α, as well as with histological examinations. We envision that the reported optical methods can be developed into a fast, real time, cost effective and easy to implement imaging approach for quantification of the browning process in adipose tissue.

Retinoic acid is abundantly detected in different depots of adipose tissue by SERS.

Retinoic acid (RA) is essential for early developmental processes and stem cell differentiation, but less is known about its contributions to adult tissues and stem cells including adipose tissue. We previously demonstrated that many genes involved in RA synthesis and downstream pathway are differentially expressed in adipose-derived stem cells (ASCs) from visceral fat compared to those from subcutaneous fat, leading to changes in their early adipogenic functions. In order to study potential contributions of RA in adipose tissue, we measured tissue RA levels using a technique based on surface-enhanced Raman spectroscopy (SERS). The data indicate heretofore underappreciated abundance of endogenous RA in mouse adipose tissue compared to other tissues and dynamic changes of RA concentrations after high fat diet feeding. Our results lay the foundation for further investigation on the functional role of RA in adipose tissue development and metabolism.

Adipocyte Ceramides Regulate Subcutaneous Adipose Browning, Inflammation, and Metabolism.

Adipocytes package incoming fatty acids into triglycerides and other glycerolipids, with only a fraction spilling into a parallel biosynthetic pathway that produces sphingolipids. Herein, we demonstrate that subcutaneous adipose tissue of type 2 diabetics contains considerably more sphingolipids than non-diabetic, BMI-matched counterparts. Whole-body and adipose tissue-specific inhibition/deletion of serine palmitoyltransferase (Sptlc), the first enzyme in the sphingolipid biosynthesis cascade, in mice markedly altered adipose morphology and metabolism, particularly in subcutaneous adipose tissue. The reduction in adipose sphingolipids increased brown and beige/brite adipocyte numbers, mitochondrial activity, and insulin sensitivity. The manipulation also increased numbers of anti-inflammatory M2 macrophages in the adipose bed and induced secretion of insulin-sensitizing adipokines. By comparison, deletion of serine palmitoyltransferase from macrophages had no discernible effects on metabolic homeostasis or adipose function. These data indicate that newly synthesized adipocyte sphingolipids are nutrient signals that drive changes in the adipose phenotype to influence whole-body energy expenditure and nutrient metabolism.

A Role for Ceramides, but Not Sphingomyelins, as Antagonists of Insulin Signaling and Mitochondrial Metabolism in C2C12 Myotubes.

The accumulation of sphingolipids in obesity leads to impairments in insulin sensitivity and mitochondrial metabolism, but the precise species driving these defects is unclear. We have modeled these obesity-induced effects in cultured C2C12 myotubes, using BSA-conjugated palmitate to increase synthesis of endogenous sphingolipids and to inhibit insulin signaling and oxidative phosphorylation. Palmitate (a) induced the accumulation of sphingomyelin (SM) precursors such as sphinganine, dihydroceramide, and ceramide; (b) inhibited insulin stimulation of a central modulator of anabolic metabolism, Akt/PKB; (c) inhibited insulin-stimulated glycogen synthesis; and (d) decreased oxygen consumption and ATP synthesis. Under these conditions, palmitate failed to alter levels of SMs, which are the most abundant sphingolipids, suggesting that they are not the primary intermediates accounting for the deleterious palmitate effects. Treating cells with a pharmacological inhibitor of SM synthase or using CRISPR to knock out the Sms2 gene recapitulated the palmitate effects by inducing the accumulation of SM precursors and impairing insulin signaling and mitochondrial metabolism. To profile the sphingolipids that accumulate in obesity, we performed lipidomics on quadriceps muscles from obese mice with impaired glucose tolerance. Like the cultured myotubes, these tissues accumulated ceramides but not SMs. Collectively, these data suggest that SM precursors such as ceramides, rather than SMs, are likely nutritional antagonists of metabolic function in skeletal muscle.