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.

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.

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.

A New Role of OmpR in Acid and Osmotic Stress in Salmonella and E. coli.

Bacteria survive and respond to diverse environmental conditions and during infection inside the host by systematic regulation of stress response genes. E. coli and S. Typhimurium can undergo large changes in intracellular osmolality (up to 1.8 Osmol/kg) and can tolerate cytoplasmic acidification to at least pHi 5.6. Recent analyses of single cells challenged a long held view that bacteria respond to extracellular acid stress by rapid acidification followed by a rapid recovery. It is now appreciated that both S. Typhimurium and E. coli maintain an acidic cytoplasm through the actions of the outer membrane protein regulator OmpR via its regulation of distinct signaling pathways. However, a comprehensive comparison of OmpR regulons between S. Typhimurium and E. coli is lacking. In this study, we examined the expression profiles of wild-type and ompR null strains of the intracellular pathogen S. Typhimurium and a commensal E. coli in response to acid and osmotic stress. Herein, we classify distinct OmpR regulons and also identify shared OmpR regulatory pathways between S. Typhimurium and E. coli in response to acid and osmotic stress. Our study establishes OmpR as a key regulator of bacterial virulence, growth and metabolism, in addition to its role in regulating outer membrane proteins.

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.

Non-canonical activation of OmpR drives acid and osmotic stress responses in single bacterial cells.

Unlike eukaryotes, bacteria undergo large changes in osmolality and cytoplasmic pH. It has been described that during acid stress, bacteria internal pH promptly acidifies, followed by recovery. Here, using pH imaging in single living cells, we show that following acid stress, bacteria maintain an acidic cytoplasm and the osmotic stress transcription factor OmpR is required for acidification. The activation of this response is non-canonical, involving a regulatory mechanism requiring the OmpR cognate kinase EnvZ, but not OmpR phosphorylation. Single cell analysis further identifies an intracellular pH threshold ~6.5. Acid stress reduces the internal pH below this threshold, increasing OmpR dimerization and DNA binding. During osmotic stress, the internal pH is above the threshold, triggering distinct OmpR-related pathways. Preventing intracellular acidification of Salmonella renders it avirulent, suggesting that acid stress pathways represent a potential therapeutic target. These results further emphasize the advantages of single cell analysis over studies of population averages.

A FRET-based DNA biosensor tracks OmpR-dependent acidification of Salmonella during macrophage infection.

In bacteria, one paradigm for signal transduction is the two-component regulatory system, consisting of a sensor kinase (usually a membrane protein) and a response regulator (usually a DNA binding protein). The EnvZ/OmpR two-component system responds to osmotic stress and regulates expression of outer membrane proteins. In Salmonella, EnvZ/OmpR also controls expression of another two-component system SsrA/B, which is located on Salmonella Pathogenicity Island (SPI) 2. SPI-2 encodes a type III secretion system, which functions as a nanomachine to inject bacterial effector proteins into eukaryotic cells. During the intracellular phase of infection, Salmonella switches from assembling type III secretion system structural components to secreting effectors into the macrophage cytoplasm, enabling Salmonella to replicate in the phagocytic vacuole. Major questions remain regarding how bacteria survive the acidified vacuole and how acidification affects bacterial secretion. We previously reported that EnvZ sensed cytoplasmic signals rather than extracellular ones, as intracellular osmolytes altered the dynamics of a 17-amino-acid region flanking the phosphorylated histidine. We reasoned that the Salmonella cytoplasm might acidify in the macrophage vacuole to activate OmpR-dependent transcription of SPI-2 genes. To address these questions, we employed a DNA-based FRET biosensor ("I-switch") to measure bacterial cytoplasmic pH and immunofluorescence to monitor effector secretion during infection. Surprisingly, we observed a rapid drop in bacterial cytoplasmic pH upon phagocytosis that was not predicted by current models. Cytoplasmic acidification was completely dependent on the OmpR response regulator, but did not require known OmpR-regulated genes such as ompC, ompF, or ssaC (SPI-2). Microarray analysis highlighted the cadC/BA operon, and additional experiments confirmed that it was repressed by OmpR. Acidification was blocked in the ompR null background in a Cad-dependent manner. Acid-dependent activation of OmpR stimulated type III secretion; blocking acidification resulted in a neutralized cytoplasm that was defective for SPI-2 secretion. Based upon these findings, we propose that Salmonella infection involves an acid-dependent secretion process in which the translocon SseB moves away from the bacterial cell surface as it associates with the vacuolar membrane, driving the secretion of SPI-2 effectors such as SseJ. New steps in the SPI-2 secretion process are proposed.

Two-component PhoB-PhoR regulatory system and ferric uptake regulator sense phosphate and iron to control virulence genes in type III and VI secretion systems of Edwardsiella tarda.

Inorganic phosphate (P(i)) and iron are essential nutrients that are depleted by vertebrates as a protective mechanism against bacterial infection. This depletion, however, is sensed by some pathogens as a signal to turn on the expression of virulence genes. Here, we show that the PhoB-PhoR two-component system senses changes in P(i) concentration, whereas the ferric uptake regulator (Fur) senses changes in iron concentration in Edwardsiella tarda PPD130/91 to regulate the expression of type III and VI secretion systems (T3SS and T6SS) through an E. tarda secretion regulator, EsrC. In sensing low P(i) concentration, PhoB-PhoR autoregulates and activates the phosphate-specific transport operon, pstSCAB-phoU, by binding directly to the Pho box in the promoters of phoB and pstS. PhoB also binds with EsrC simultaneously on the promoter of an E. tarda virulence protein, evpA, to regulate directly the transcription of genes from T6SS. In addition, PhoB requires and interacts with PhoU to activate esrC and suppress fur indirectly through unidentified regulators. Fur, on the other hand, senses high iron concentration and binds directly to the Fur box in the promoter of evpP to inhibit EsrC binding to the same region. In addition, Fur suppresses transcription of phoB, pstSCAB-phoU, and esrC indirectly via unidentified regulators, suggesting negative cross-talk with the Pho regulon. Physical interactions exist between Fur and PhoU and between Fur and EsrC. Our findings suggest that T3SS and T6SS may carry out distinct roles in the pathogenicity of E. tarda by responding to different environmental factors.

Crystal structure of the heteromolecular chaperone, AscE-AscG, from the type III secretion system in Aeromonas hydrophila.

BACKGROUND: The putative needle complex subunit AscF forms a ternary complex with the chaperones AscE and AscG in the type III secretion system of Aeromonas hydrophila so as to avoid premature assembly. Previously, we demonstrated that the C-terminal region of AscG (residues 62-116) in the hetero-molecular chaperone, AscE-AscG, is disordered and susceptible to limited protease digestion. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report the crystal structure of the ordered AscG(1-61) region in complex with AscE at 2.4 Å resolution. Helices α2 and α3 of AscE in the AscE-AscG(1-61) complex assumes a helix-turn-helix conformation in an anti-parallel fashion similar to that in apo AscE. However, in the presence of AscG, an additional N-terminal helix α1 in AscE (residues 4-12) is observed. PscG or YscG in the crystal structures of PscE-PscF-PscG or YscE-YscF-YscG, respectively, assumes a typical tetratricopeptide repeat (TPR) fold with three TPR repeats and one C-terminal capping helix. By comparison, AscG in AscE-AscG(1-61) comprises three anti-parallel helices that resembles the N-terminal TPR repeats in the corresponding region of PscG or YscG in PscE-PscF-PscG or YscE-YscF-YscG. Thermal denaturation of AscE-AscG and AscE-AscG(1-61) complexes demonstrates that the C-terminal disordered region does not contribute to the thermal stability of the overall complex. CONCLUSION/SIGNIFICANCE: The N-terminal region of the AscG in the AscE-AscG complex is ordered and assumes a structure similar to those in the corresponding regions of PscE-PscG-PscF or YscE-YscF-YscG complexes. While the C-terminal region of AscG in the AscE-AscG complex is disordered and will assume its structure only in the presence of the substrate AscF. We hypothesize that AscE act as a chaperone of the chaperone to keep AscG in a stable but partially disordered state for interaction with AscF.

Structural basis for the secretion of EvpC: a key type VI secretion system protein from Edwardsiella tarda.

The recently identified type VI secretion system (T6SS) is implicated in the virulence of many gram-negative bacteria. Edwardsiella tarda is an important cause of hemorrhagic septicemia in fish and also gastro- and extra-intestinal infections in humans. The E. tardavirulent protein (EVP) gene cluster encodes a conserved T6SS which contains 16 open reading frames. EvpC is one of the three major EVP secreted proteins and shares high sequence similarity with Hcp1, a key T6SS virulence factor from Pseudomonas aeruginosa. EvpC contributes to the virulence of E. tarda by playing an essential role in functional T6SS. Here, we report the crystal structure of EvpC from E. tarda PPD130/91 at a 2.8 Å resolution, along with functional studies of the protein. EvpC has a ß-barrel domain with extended loops. The ß-barrel consists of 11 anti-parallel ß-strands with an α-helix located on one side. In solution, EvpC exists as a dimer at low concentration and as a hexamer at higher concentration. In the crystal, the symmetry related EvpC molecules form hexameric rings which stack together to form a tube similar to Hcp1. Structure based mutagenesis revealed that N-terminal negatively charged residues, Asp4, Glu15 and Glu26, and C-terminal positively charged residues, Lys161, Lys162 and Lys163, played crucial roles in the secretion of EvpC. Moreover, the localization study indicates the presence of wild type EvpC in cytoplasm, periplasm and secreted fractions, whereas the N-terminal and C-terminal mutants were found mostly in the periplasmic region and was completely absent in the secreted fraction. Results reported here provide insight into the structure, assembly and function of EvpC. Further, these findings can be extended to other EvpC homologs for understanding the mechanism of T6SS and targeting T6SS mediated virulence in gram-negative pathogens.

Temperature and Mg2+ sensing by a novel PhoP-PhoQ two-component system for regulation of virulence in Edwardsiella tarda.

The PhoP-PhoQ two-component system is commonly used by bacteria to sense environmental factors. Here we show that the PhoP-PhoQ system of Edwardsiella tarda detects changes in environmental temperature and Mg(2+) concentration as well as regulates the type III and VI secretion systems through direct activation of esrB. Protein secretion is activated from 23 to 35 °C or at low Mg(2+) concentrations, but it is suppressed at or below 20 °C, at or above 37 °C, or at high Mg(2+) concentrations. The effects of temperature and Mg(2+) concentration are additive. The PhoQ sensor domain has a low T(m) of 37.9 °C, and it detects temperatures through a conformational change of its secondary structure. Mutation of specific Pro or Thr residues increased the stability of the PhoQ sensor drastically, altering its temperature-sensing ability. The PhoQ sensor detects Mg(2+) concentration through the direct binding of Mg(2+) to a cluster of acidic residues (DDDSAD) and through changes that likely affect its tertiary structure. Here, we describe for the first time the use of PhoP-PhoQ as a temperature sensor for bacterial virulence control.