However, an appreciation of the truly heterogeneous nature of this unique stem cell group has identified a need to more accurately delineate subpopulations by any of a host of methods, to include functional properties or surface marker expression. Cells selected for improved proliferative, differentiative, angiogenic or ischemia-resistant properties are but a few attributes that could prove beneficial for targeted treatments or therapies. Optimizing cell culture conditions to permit re-introduction to patients is critical for clinical translation.We have witnessed a rapid expansion of  Significant scientific grounding in translational stem cell research combined with the more recent expansion of adipose tissue plasticity has identified adipose-derived stem cells (ASCs) as an important tool in regenerative medicine. However, building on lessons learned in other stem cell modalities, the need to characterize cell populations appears to be critical to enable selection of those groups that will maximize laboratory and potentially clinical outcomes. More accurate delineation of ASC subpopulations is also likely to be critical to patient safety where ASC augmentation is envisaged in a clinical context. A natural precedent for investigating ASC use in reconstructive surgery involving adipose tissue has already been set; however, it appears that ASCs may play an important role in translational research in a spectrum of other clinical specialties.At a cellular level, adipose tissue consists of mature adipocytes surrounded by fibroblasts, nerves, endothelial cells, immune cells and preadipocytic cells contained within a stromovascular cell network . EnzymatOrigins of adipose tissue can be traced to the embryonic mesoderm, from where mesenchymal stem cells (MSCs) possess the ability to differentiate not only into adipocytes, but also chondrocytes, myocytes, osteoblasts  or indeein vitro and even stringent isolation protocols for enriched HSCs can produce cells that do not demonstrate pluripotency or self-renewal. Within bone marrow, HSCs are surrounded by supporting bone marrow stromal cells (BMSCs) [Ongoing ethical dilemmas and concerns over oncogenesis in patients have restricted clinical use of embryonic stem cells ; however (BMSCs) , a subpo (BMSCs) . Such na (BMSCs) .in vitro [Despite numerous studies attempting to define MSCs by site, surface markers or differentiation capacity, a standard accepted definition is lacking. The International Society for Cellular Therapy (ISCT) has previously defined three pre-requisites for consideration of a MSC: plastic adherence in standard culture conditions; expression of cell surface markers CD105, CD73, CD90 and lack of expression of CD45, CD34, CD14 or CD11b, CD79α, CD19, HLA-DR; and ability to differentiate into osteoblasts, adipocytes and chondroblasts in vitro . Of partin vitro .in vitro and without the ethical concerns attributed to embryonic stem cells raise the attractiveness of the MSC for clinical applications; bone, liver, cardiac, skeletal muscle and CNS applications are at various stages of translation, including clinical trials [Relative ease of expansion by well-described protocols, ability for induced-differentiation into a host of cell lines l trials .5 MSCs in culture adhere after initial plating [Investigation and clinical use of BMSC is now standard; however, concerns over acceptability of harvesting techniques and potentially low cell yields  have plating . Charact plating , while b plating .in vitro differentiation to adipogenic, chondrogenic, osteogenic and even neural end states has been described, confirming multilineage capacity and application of the term, ASCs. This is important to distinguish from the relatively heterogeneous population of SVF cells present after immediate tissue digestion by collagenase. Lack of uniformity in cellular characterization is likely a key contributing factor to the many and varied terminologies applied to these populations [A catalog of studies attempting to characterize this heterogeneous cell population have followed , with coulations : PLA celulations .in vitro from SVF cells over multiple passages have also been noted [Temporal changes en noted ,23. The en noted .Although some degree of conformity now exists in recognition of a baseline ASC phenotype within SVF, the presence and functional role of numerous other markers not classically associated with MSCs has become established as a rapidly expanding niche within the adipose stem cell arena. Subpopulations selected for improved proliferative, differentiative, angiogenic or ischemia-resistant properties are promising attributes that may prove to optimize targeted treatments or therapies.The precise location of stem cells within native tissue is of significant interest, not only for aiding processing and culture within the laboratory, but also for recognizing that progenitor cells from different sources exhibit similar lineage differentiation properties in the same environment. In fact, there is considerable evidence that MSCs may be derived from a common perivascular origin. Culture of perivascular cells from multiple tissues results in products expressing CD surface markers typical of MSCs  that exhibit anticipated clonal proliferation and multilineage potential in suitable inductive conditions .+ cells as being closely associated with vessels. Although small numbers of these are CD31+ and likely capillary endothelial cells, predominant perivascular CD34+/CD31− populations are typical of ASCs and presumably pericytic. In vitro separation of CD34+/CD31−/CD144− ASCs from endothelial cells (CD34+/CD31+/CD144+) by differential plastic adherence further supports distinct populations, with the majority of the ASC subset co-expressing MSC, pericytic and smooth muscle markers [Specific examination by several authors appears to corroborate these data for ASCs. Immunofluorescence of adipose tissue sections identifies populations of CD34 markers .−/CD34−/CD45−/CD146+, while ‘supra-adventitial-adipose stromal cells’ localizing to the outer aspect of the vascular ring were characterized as CD31−/CD34+/CD45−/CD146− cells. Both populations demonstrated adipogenesis, although the smaller-numbered pericytes more convincingly so [Other studies have employed immunohistochemistry techniques in combination with flow cytometry to further define population subsets  based on direct relation to vessels. Pericytes, constituting the innermost layer of stromal cells contacting vessel endothelium, were described as CD31ingly so .in vivo and in vitro studies discussed later in this review employ populations matching, either in part or completely, these population subsets.Of note is the crossover between populations defined as pericytic or perivascular and those defined as ASC. As an example, a ‘supra-adventitial’ population phenotype  is identin vivo [Considering various anatomical depots, important differences have been noted in number and properties of harvested ASCs. Some authors have suggested the abdomen to have higher ASC yield than the hip/thigh, although found no difference in differentiation capacity between the two sites (for chondrogenic and osteogenic lineages) . Others in vivo .Depth of adipose tissue harvest also appears to be critical to function, with ASCs derived from subcutaneous fat demonstrating more rapid growth and adipogenesis than those from visceral fat . CompariMerits of selecting for surface markers expressed by ASCs are now well-recognized and a focus of investigation for several groups. Comparisons of sorting methods employed for subpopulation isolation are beyond the scope of this review, but largely encompass magnetic- or fluorescence-activated cell-sorting techniques.Understanding overlap and significant differences in adipose-derived populations from bone marrow or hematopoietic lines has gathered interest. CD34, initially assumed hematopoietic-only marker, has seen a plethora of recent investigative studies . Demonst+ versus CD34− populations selected by flow cytometry has been demonstrated reproducibly [− cells are described to contribute a more adipogenic and osteogenic cell lineage than positively selected cells [+/CD31− represent a preferentially adipogenic subpopulation in stimulatory culture conditions and therefore likely contain key preadipocyte fractions. The absence of hematopoietic colony formation in stimulatory assays may refute the suggestion of CD34 as a hematopoietic-only marker that is present in digested adipose tissue only as a consequence of residual circulating blood; however, the presence of CD34 is widespread in many other tissues and this must also be considered [Furthermore, improved proliferation in CD34oducibly ,36. The nsidered .+ selected cells [The impact of co-culturing ASCs with mature adipocytes has been studied, to mimic the native environment within a transplanted fat graft and also the simulated ASC-augmented graft. Models employing subpopulations, specifically CD34, are described. Co-culture conditions resulted in significantly faster proliferation versus both standard and adipogenic medium for CD34ed cells .+/CD90+ populations are not constrained to adipogenic properties alone, but also show endothelial cell differentiation with high VEGF production from capillary-like structures [CD34ructures . Subpopu− cells [+ ASC, multilineage differentiation and improved proliferative rates have been observed when compared with unsorted plastic adherent cells and CD34+ populations [Establishing more detailed temporal alterations in expression may allow for optimal subpopulation selection. Indeed, data have suggested early expression of CD34 following initial ASC attachment in culture, followed by upregulation of CD105, CD146, CD271 and subsequent loss of CD34 expression . Further− cells . Buildinulations .+ cells show significantly greater chondrogenic potential in vitro, confirmed on tissue-culture plastic, gel-embedded sheets [The molecule CD105 (endoglin) is believed to have an intrinsic role in cellular attachment, aided by both CD146 and CD271 . Positivd sheets  and biodd sheets . Scope fd sheets .+ cells treated with a standard induction regimen (5-azacytidine) show significantly greater cardiomyocyte differentiation versus CD73- cells, as determined by myofibril and cardiac surface marker demonstration [+ selection has been extended further afield to generate subpopulations capable of albumin production and ammonia detoxification, mimicking the role of primary human hepatocytes and offering hope to use of ASCs in regenerative hepatobiliary medicine [As the markers CD73, CD90 and CD105 are ISCT-defining of MSCs, it is logical they may characterize cells within an early undifferentiated state and thus offer greater potential for widespread tissue differentiation. To consider specific examples, CD73stration . Mean whmedicine .+ adipogenic and osteogenic differentiation versus mixed (unsorted) cell populations, has been shown. Interestingly, decreased expression in culture may indicate SSEA-4 as a relatively early, rather than late, MSC marker [+ subpopulations have been demonstrated to progress along osteogenic and endothelial cell lineages, raising possibilities for tissue engineering translation, for example, vascularized bone constructs [Despite the widespread acceptance of characterizing MSC and subsequently ASC populations by CD markers, allowing some element of conformity among researchers, it is recognized these are unlikely to represent a definitive phenotype. Identification of embryonic and pluripotent stem cell markers within adipose-derived cell populations lends weight to the argument that a plethora of markers await exploration. SSEA-4, an early-expressed embryonic glycoprotein demonstrated in embryonic stem cells, marrow-derived MSCs and numerous oncological cell lines, is one such marker. High prevalence in human breast  adipose tissue, with improved SSEA-4C marker . In apprnstructs .in vitro characterization of ASC subpopulations offers insight into potential gain from selection; however, experience of translation to the clinical arena in medical research has warned us that changes observed on tissue-culture plastic may correlate poorly with those seen in the patient. Optimization of in vivo models is therefore critical to bridging the link from culture to clinic.The in vivo. Nevertheless, a steady influx of data pertaining to a breadth of tissue types is being seen.In comparison to the broader entity of stem cells, ASCs and particularly subpopulation selection remain in relative infancy with respect to quantity and quality of reproducible experimental data +/CD146−/CD31− cells present in SVF (defined as ‘ASC yield’) has been shown to directly correlate to fat graft volume retention in nude mice when small volumes are injected subcutaneously  of CD34+/CD90+ seeded-constructs were confirmatory of observed macro- and micro-scopic adipocyte differentiation not observed with unsorted controls [Of the numerous studies discussed, many have utilized CD34 as a marker of proliferation and indeed plastic adherence. Further refinement within CD34 subpopulations by positive (rather than negative) co-selection of other markers may be of benefit in producing cells with targeted properties. Building on and confirming previous work demonstrating improved proliferation of CD34controls . The tru+ cells from within a murine Lin−CD29+CD34+Sca-1+CD24+ population that constituted only 0.08% of total SVF cells. Subsequent injection of GFP-labeled CD24+, CD24− or unsorted cells into A-zip lipodystrophic mice demonstrated that only CD24+ cells were able to reconstitute adipose depots in vivo [− cells in vivo, from within a day after seeding. Given established deficits of CD24− subpopulations forming adipose depots, it is likely that CD24+ adipocyte progenitors give rise to CD24− cells, which may be further differentiated toward mature adipocytes [in vitro proliferative and adipogenic capacity, may not in fact correlate to reciprocated in vivo results. Accurate mapping of temporal changes in expression following transplantation of a subpopulation may therefore be essential to dissecting the pathway from cell sorting to generated functional tissue.Other studies have examined properties of subpopulations present in remarkably low quantities within SVF, including the use of CD24 in vivo . Later wipocytes . Thus, sin vitro data discussed above, has been utilized for adipocyte generation following subcutaneous injection of GFP-labeled ASCs loaded onto fibrin constructs [Finally CD271, with robust multilineage capacity from nstructs . Confirmin vivo has been observed for a host of other surface markers, including MSC markers that have shown in vitro promise. CD90+-enriched ASCs have recently been shown to undergo improved osteogenic differentiation over CD90−, CD105+ and unsorted cells. Results were corroborated by increased bone formation in CD90+ groups when seeded on hydroxyapatite-coated polylactic-co-glycolic acid scaffolds for the repair of calvarial defects in mice [Translation  in mice .+ populations has been suggested elsewhere. Previously discussed in vitro data support CD105+ as a highly osteogenic subpopulation [high+ and CD105low+), demonstrating CD105low+ subpopulations constitute the more osteogenic population in vitro and in vivo. Significant differences were confirmed by serial micro-CT scanning of mice with parietal bone defects treated with CD105low+ cells on hydroxyapatite-coated polylactic-co-glycolic acid scaffolds, versus CD105high, unsorted and scaffold-only groups [low+/CD90high+ lends to a more osteogenic phenotype than CD105low+/CD90low+in vitro; however, due to this population representing only 16% of ASCs, the authors excluded it from in vivo work on the premise that clinical translation was unlikely. This may support the role of CD90 in osteogenic differentiation as previously suggested [Despite these findings, the osteogenic role of CD105pulation . Other sy groups . The latuggested ; howeverin vivo transplantation. However, cellular interaction in an animal model is inherently difficult to predict. Specific subpopulations within ASCs may be pre-defined to descend a particular lineage, which is only accelerated by the use of pre-induction protocols. Similarly, if the host microenvironment is the dictating factor in subpopulation behavior, then perhaps selection would occur regardless of intervention.In the majority of studies discussed, pre-induction of ASCs in media specific for an end-target is routine prior to +/CD73+/CD90+/CD105+, unsorted cells subject to osteogenic differentiation media prior to experimental seeding (pre-induced unsorted) and pre-induced cells that had been further sorted for the above CD markers (pre-induced sorted). Of particular note, despite improved osteogenic capacity of pre-induced cells in vitro, after 8 weeks of in vivo seeding on β-tricalcium phosphate scaffolds, there were no significant differences in area of new bone formation relative to noninduced cells. Furthermore, cell sorting for any of the studied markers also failed to identify any differences (with or without pre-induction) [A recent study has compared osteogenesis among four different ASC populations: unsorted cells, cells sorted for CD44duction) . On the in vivo bone regenerative capabilities. Regular detection in early characterization studies  [CD105 is not alone in being pursued as a translational osteogenic marker; several others including Stro-1 have been selected for assessment of  studies  has convstructs)  and at lstructs) .−CD271+Sca-1+) have also been employed for osteoblast formation within biphasic calcium phosphate-scaffolds in vivo.Furthermore, CD271 population subsets generating adipocytes above (Lin+/NG2−) have myogenic differentiation properties in vivo following seeding on cross-linked hyaluronic acid scaffolds in mice. However, CD34+/NG2− cells lack all myogenic capabilities, but may reliably differentiate into adipocytes [Further demonstration of the effect of selecting more than one cell-surface marker can be seen with the pericyte marker neuron-glial 2 (NG2). Cells selected for CD34 and NG2  livers with improvement of basic liver functions occurs [Transdifferentiation potential of CD105s occurs .in vitro subpopulation selection have undergone in vivo transition to date, although the breadth of data available (even within the same surface marker) creates difficulty in easily identifying the specific marker(s) most likely to have a desired end-function. However, the accelerated interest in implementation of ASCs, largely utilizing unsorted populations, has already become established across a number of clinical specialties.Thus, it can be seen that a significant number of surface markers highlighted for ASC populations employed clinically must be defined clearly by degree of cellular manipulation prior to patient re-introduction. Broadly, this encompasses unprocessed SVF cell populations (by definition not ASCs), unsorted and sorted ASCs. Debate over the correct nomenclature ascribed to cells has continued for some time, but it is the isolated, relatively homogenous, plastic-adherent multipotent precursor cells identified from human adipose tissue only that may correctly be termed ASC, as discussed above .et al. expanded on their earlier work characterizing surface markers in lipoaspirate samples and adherent-ASCs [Within the field of reconstructive surgery, the largest case series employing precursor cells from adipose tissue have been in the treatment of patients undergoing cosmetic breast procedures. In 2008, Yoshimura ent-ASCs  to releaent-ASCs .Later work employing the CAL technique to address volume deficiency after implant removal in 15 patients who had developed capsular contracture reported a 40–80% graft take as determined by 3D photographic assessment. No complications  were detected on postoperative MRI or mammography . The autAlso falling within a reconstructive remit, ASCs have been employed for the treatment of end-stage radiation tissue necrosis in breast cancer patients, although the authors used non-PLA assumed to contain ASCs without SVF separation or culture .ex-vivo culture is potentially appealing, although a true appreciation of absolute cell yield or composition is extremely difficult to ascertain. In a series of 67 patients who had undergone surgery to remove breast cancer but to preserve the unaffected breast tissue, the use of ‘ADRC-enriched’ fat grafts for reconstruction of the breast tissue defect resulted in high satisfaction levels reported by clinicians and patients alike [in vivo interaction, the question arises whether further basic scientific grounding is required before any future clinical introduction.In order to facilitate ‘bedside’ ASC-integration, groups have employed the use of specifically manufactured commercial products that utilize enzymatic digestion to deliver ‘adipose-derived regenerative cell-enriched grafts’. Negating the need for transport of tissue and in vitro and in vivo model) determined that ASCs do augment the growth of active, but not resting breast cancer cells. The authors stated that extrapolation of these results may suggest the potential for ASCs to promote breast tissue regeneration but should not affect the status of dormant residual cancer cells [in vivo studies have identified increased proliferation of tumor cell lines, in both co-culture with ASCs and also when treated with ASC-conditioned media. Despite no increase in new tumor vessel formation, a decreased rate of apoptosis in ASC presence may suggest preference toward tumor growth in the ASC-supplemented environment [One of the few studies examining effects of ASCs on breast cancer cells  on various tumor sizes, growth and metastases outlined the difficulties in ascertaining safety even at a preclinical stage. With a catalog of data suggesting that MSCs may promote or alternatively inhibit tumor growth, the authors correctly conclude that our current knowledge of mechanisms by which MSCs may exert their effects is still poorly understood, such that predictions on cell behavior and anticipated safety cannot be made. The authors do stress that there has been no evidence of tumor formation directly attributed to MSC use in all patients treated to date . Clearlyin vitro prior to supplementation in subcutaneously injected fat grafts, albeit in healthy nononcological cohorts. A concentration of 20 × 106/ml ASCs employed to enrich grafts resulted in significantly higher residual volumes at a 121-day end point (80.9 vs 16.3% in nonsupplemented control grafts), confirmed by serial MRI imaging and surgical removal of grafts at experimental completion [Regardless, we have now seen the first landmark clinical trial of unsorted ASCs cultured mpletion . The usempletion ) and relde novo vascularized bone flap for palatal reconstruction in craniofacial surgery [Aside from use as adjuncts to autologous fat grafting, expanded ASCs have also been employed in colorectal surgery for perianal fistula treatment since 2003 , with re surgery .in vitro and now in vivo, the final bridging step to ASC selection for use in the clinical arena now appears to be on the horizon.Rapid expansion of interest in ASC translation is reflected in the breadth of specialties for which patients are currently being recruited. A recent search under the criteria ‘adipose stem cell’ reveals 109 clinical trials currently registered with the US Institute of Health , a marke+ content within stem cell transplants is widely used as a predictor of graft success and large doses of CD34+ purified peripheral blood cells have been administered to HLA-mismatched patients on the premise of selection-induced T-cell depletion with subsequently reduced risk of graft-versus-host disease [Although clinical ASC subpopulation data are scarce, other sources of adult stem cells, particularly hematopoietic populations, have seen tremendous influx of subpopulations employed clinically. CD34 disease . The lit+ data may also be found in cardiac research, with Phase II trials of CD34+ intramyocardial infusion into infarcted tissue already completed. Reduction in angina frequency and improvement in exercise tolerance have been reported [+ populations have also been directly compared via transendocardial versus intracoronary infusion techniques in the treatment of dilated cardiomyopathy, with the former correlating to improved left ventricular function and exercise capacity [in vivo data, leukapheresis samples from patients with liver insufficiency have been isolated for CD34 using commercially available magnetic sorting techniques and subsequently infused under image guidance directly into the portal vein or hepatic entry [Significant CD34reported , with Phreported . Purifiecapacity . Mirroriic entry . The safic entry .Should adipose tissue-based research progress in the manner observed for hematopoietic work, we are likely to see an expansion in ASC subpopulation trials pending clinical introduction. For this to be a measured reality, certain aspects of processing, culture (where applicable) and patient re-introduction of ASCs demand refinement.ex vivo expansion, involves numerous steps, which individually and cumulatively have direct potential for patient harm. Strict regulation in the transfer and use of tissue is required to minimize risk. Sterility is paramount at all times, but particularly when laboratory processing and culture is undertaken. The increased implementation of commercial products that allow bedside subpopulation sorting prior to immediate use ‘on-table’ is suggested to reduce these risks; however, in return the ability to characterize and expand populations is lost. Of course, the logistical and financial effects of reducing patient re-operation should not be understated, nor the potential impact on patient satisfaction, all of which must be counterbalanced by significant start-up and training costs.Current use of ASCs, with or without Where adipose tissue is removed from a clinical setting, assuming sterile handling and appropriate transportion methods, it is the cellular digestion and culture of tissue that introduces the greatest variables to the regenerative loop that ultimately returns to the patient. Protocols for ASC harvesting are well described and almost universally adopted. Of particular note is the use of xenogenic animal-derived products that introduce the risk of contamination and prompt serious biosafety considerations. Transfer of bacterial or viral infections , prions The use of collagenase for adipose tissue digestion prior to SVF isolation is widely adopted. However, a number of current collagenase products available are not clearly defined to be devoid of animal products, therefore by definition may be xenogenic. Animal-free collagenase products are available, but their current use tends to be limited and may be prohibited by cost. When establishing alternatives or substitutes to any given product, unchanged cellular morphology, phenotype (to include surface marker characterization) and behavior in culture must be demonstrated. A recent study comparing research-grade collagenase  with ‘animal origin-free’ and xeno-free lyophilisates established no differences in cell yield, proliferation, CD marker phenotype or differentiative capacity .ex vivo expansion of ASCs with clinical intent remains the identification of xeno-free alternatives to standard FBS-based media, devoid of bovine products and with reduced lot-to-lot variability. Numerous options have been considered for the culture of bone-marrow-derived MSCs; however, data concerning ASCs have been more limited. As the regenerative potential of ASCs now becomes more widely recognized, a parallel surge in clinical translatability studies is being seen.A further challenge in Significantly improved ASC proliferation with retention of multilineage capacity has been demonstrated in commercially available serum-free xeno-free media ,92, althMost recently, authors have recognized the need for complete xeno-free protocols, with the use of human serum, human serum albumin and Tryple as alternatives to FBS and trypsin in the culture of ASCs . ImproveA host of other serum alternatives have been suggested for unique benefits, reduced immunogenicity using umbilical-cord-derived extracts as an example . Prolifeex vivo ASC expansion.Serum-free alternatives now command significant interest from the scientific and clinical community alike; however, the body of experimental data relating to ASCs still remains based on bovine serum. Each of the suggested products has merits and disadvantages, which are reviewed in extensive detail individually elsewhere. In general terms, the ideal culturing media should be cost effective, have minimal batch variability, low immunogenicity and be free of all animal, but also likely allogenic derivatives. Perhaps in time we will see the synthetic serum-free xeno-free product become the frontrunner in in vitro and in vivo models, before what may be potentially hasty progression to patient administration.Within the last decade, a great volume of laboratory data targeting unsorted ASCs to a spectrum of clinical problems has seen rapid escalation to clinical trials. However, we must not lose sight of the importance of adequate scientific grounding in both Within ASC research, the identification, characterization and selection of subpopulations have identified unique challenges with potentially even greater translational benefit. While the evidence for specific ASC selection as a therapy toward specific clinical scenarios is yet to be determined, this is likely to represent an important component of clinical advancement within this field over the next 5 years. Reconstructive surgery, cardiac, musculoskeletal and neurological medicine are a few of the areas that may profit.ex-vivo expansion. For the latter, stringent adherence to good clinical practice and laboratory principles alike will be fundamental to ensure optimum patient outcomes.Further well-conducted, reproducible data are required that will answer numerous questions, not least between immediate population sorting and re-introduction over


Errors in sample annotation or labeling often occur in large-scale genetic or genomic studies and are difficult to avoid completely during data generation and management. For integrative genomic studies, it is critical to identify and correct these errors. Different types of genetic and genomic data are inter-connected by cis-regulations. On that basis, we developed a computational approach, Multi-Omics Data Matcher (MODMatcher), to identify and correct sample labeling errors in multiple types of molecular data, which can be used in further integrative analysis. Our results indicate that inspection of sample annotation and labeling error is an indispensable data quality assurance step. Applied to a large lung genomic study, MODMatcher increased statistically significant genetic associations and genomic correlations by more than two-fold. In a simulation study, MODMatcher provided more robust results by using three types of omics data than two types of omics data. We further demonstrate that MODMatcher can be broadly applied to large genomic data sets containing multiple types of omics data, such as The Cancer Genome Atlas (TCGA) data sets. Many human diseases are complex with multiple genetic and environmental causal factors interacting together to give rise to disease phenotypes. Such factors affect biological systems through many layers of regulations, including transcriptional and epigenetic regulation, and protein changes. To fully understand their molecular mechanisms, complex diseases are often studied in diverse dimensions including genetics (genotype variations by single nucleotide polymorphism (SNP) arrays or whole exome sequencing), transcriptomics, epigenetics, and proteomics. However, errors in sample annotation or labeling often occur in large-scale genetic and genomic studies and are difficult to avoid completely during data generation and management. Identifying and correcting these errors are critical for integrative genomic studies. In this study, we developed a computational approach, Multi-Omics Data Matcher (MODMatcher), to identify and correct sample labeling errors based on multiple types of molecular data before further integrative analysis. Our results indicate that signals increased more than 100% after correction of sample labeling errors in a large lung genomic study. Our method can be broadly applied to large genomic data sets with multiple types of omics data, such as TCGA (The Cancer Genome Atlas) data sets. Cells employ multiple levels of regulation that enable them to respond to genetic, epigenetic, genomic, and environmental perturbations. With advances in high-throughput technologies, comprehensive data sets have been generated to measure multiple aspects of biological regulation, such as genetics, transcriptomics, metabolomics, glycomics, and proteomics. To elucidate the complexity of cell regulation, diverse types of data from these different technologies must be integrated.Sample errors, including sample swapping, mis-labeling, and improper data entry are inevitable during large-scale data generation. Some of these errors can be detected during quality control (QC) on each type of data; however, others are more elusive and may affect integrative data analysis, depending on the integration methods used. In some integrative analyses, signature sets are first defined by each data type individually, for example signatures for gene expression, methylation, or copy number variation (CNV). Then, the signatures are overlapped to identify high-confidence changes To fully understand biological systems, it is necessary to elucidate how genetic and epigenetic perturbations lead to transcriptomic and proteomic changes, which in turn contribute to the disease phenotype. Simultaneously considering different types of biological data can result a better understanding of biological systems http://research.mssm.edu/integrative-network-biology/Software.html.With recent advances in high-throughput technologies, multiple layers of molecular phenotypes have been measured in the same sample for comprehensive survey of biological systems. To maximally utilize these data, it is necessary to properly match different types of data pertaining to the same sample or individual before integrative analyses. Here we present a sample mapping procedure called Multi-Omics Data matcher (MODMatcher), which not only identifies mis-matched omics profile pairs, but also properly assigns them to the correct samples based on other omics data. We applied MODMatcher to two large-scale public multi-omics datasets: one from the Lung Genomic Research Consortium (LGRC) and one from The Cancer Genome Atlas (TCGA). In both cases, adjustment for mis-matched samples improved data consistency and increased statistic power to identify biological regulations. All software programs and scripts are available at http://www.lung-genomics.org). Genotype data was provided by the LGRC consortium. The data set consists of gene expression profiles of lung tissues from 219 patients with COPD and 108 non-disease controls (CTRL), and methylation profiles of lung tissues from 173 COPD patients and 76 controls. First, the gender of each sample was inferred based on three types of data and compared to the gender annotated in clinical data. There was no ambiguity in gender prediction based on each individual type of data; the molecular profiles of different genders were clearly separated  for gene expression profiles (FAM197Y2P (see Methods), we found 1 gender inconsistency in CTRL samples and 15 in COPD samples, corresponding to a mismatch error rate of 6.4% (16/249) for methylation profiles (The LGRC is a consortium for studying chronic lung diseases including chronic obstructive pulmonary disease (COPD). Clinical information and gene expression and methylation profiling data were obtained from the LGRC data portal  resulted in better alignment than duo alignments (considering two types of data at a time) combined. From the sample alignment of the LGRC data as describe above, we identified 76 COPD samples with aligned genotype, gene expression and methylation profiles. Among these 76 samples, only 65 could be correctly matched when individual similarity scores such as cis mRNA-methylation pairs were used. For a fair comparison of trio and duo alignment, we simulated sample labeling errors by randomly assigning sample labels using only these 65 COPD samples. As in the empirical data, we kept low error rates in SNP and gene expression profiling data. We increased the number of mis-labeled methylation profiles from 0 to 24 (corresponding error rate 0% to 37%). At each error rate, we simulated 5 independent data sets and used the average for comparison. In both of duo and trio alignment, all three data types were used but in different ways. In duo alignment, we identified the sample pairs from each pair of data types independently and summarized them to have final pairs. For an example, a methylation profile can be matched with an mRNA profile directly based on the identity similarity score ere high . Trio alere high . As erroK-01”, and “TCGA-BH-A18T-01”, were cross-aligned to each other in methylation and gene expression profiles  is the first cancer data set in TCGA consisting of CNV, gene expression, and methylation profiles. There were 470 GBM tumor samples with both CNV and mRNA profiles. We identified 24 mis-aligned profile pairs. Two of them were cross-aligned between CNV and gene expression profiles  . When weblematic . Additioblematic . These fIn large-scale genetic and genomic studies, errors in sample annotation or labeling are common and difficult to avoid completely. Identifying and correcting these errors is critical for statistical analysis, especially for integrative analysis. In this study, we introduce an iterative computational procedure, MODMatcher, that uses multiple types of molecular data  for sample alignment by using cis regulation pairs of each pair of data types to calculate sample identity similarity scores. When applied to two large public data sets, LGRC and TCGA, MODMatcher not only identified mis-aligned profile pairs but also corrected and rescued mis-labeled samples. In a simulation study of COPD samples in the LGRC set, sample alignment with three types of data (trio matching) performed better than alignment with two types of data (duo matching). When applied to the GBM data set in TCGA, trio matching unambiguously identified the source of sample labeling errors. Thus, MODMatcher can rescue mis-aligned or mis-labeled samples to maximize statistical power in integrative analysis in large-scale genetic and genomic studies. Indeed, correction of mis-aligned samples increased both the number of cis pairs identified and the statistical significance.Sample labeling errors are not unique to a few data sets, but are inevitable for any large data sets, despite intensive efforts in QCing each type of data individually. Our methods based on methylation profiles for gender inference and alignment with other omics profiles are novel and have not been included in standard data QC procedures. We applied our methylation-based gender inference method to more TCGA data sets and demonstrated that gender can be unambiguously inferred from methylation profiles . We idenIt is worth to note that the sample identity similarity scores, MODMatcher has several features not found in existing sample alignment methods such as MixupMapper http://genenetwork.nl/wordpress/mixupmapper/#additional) and compared alignment results based on the two methods . The genotypic data was provided by the LGRC consortium. The gene expression data was generated with Agilent V2 human whole genome arrays. The processed mRNA array data was downloaded from the LGRC website. DNA methylation data was generated with Nimblegen 2.1M Whole-Genome Tiling Arrays. Raw DNA methylation data was downloaded from the LGRC website. The quality of each probe was compared with background probe signals, and probes with low quality were excluded from further analysis. Then DNA methylation level  of each tiling probe was estimated with the CHARM method Clinical, gene expression and methylation data were obtained from the LGRC data portal (https://tcga-data.nci.nih.gov/tcga/). To illustrate our sample alignment procedure, we selected BRCA (one of the newest cancer data sets) Different types of clinical and molecular data of various cancers are publicly available at the TCGA data portal  is highly expressed in male Gene expression levels of Y-chromosome specific genes can also be used to reliably predict gender information. FAM197Y2P, TTTY15, and TBL1Y were significantly associated with genders in the LGRC data set . A methyl probe, “chrY:9994006”, representing FAM197Y2P is the best methyl probe for gender prediction and was used to classify samples in the LGRC data set into male and female. Raw intensity data in methylation profiling was used to determine whether probes mapped to Y-chromosome DNA fragments can be used to classify samples into male and female. Raw intensities of probes representing the Y-chromosome specific genes An eSNP is a single nucleotide polymorphism (SNP) whose genotype associates with variation in the expression of a particular gene. If that gene and its corresponding eSNP are in proximity, the eSNP is called a cis-eSNP. Cis-eSNPs have been extensively studied for their association with disease risks i. Second, genotype at the cis-eSNP for sample i is inferred by comparing its gene expression level i at the cis-eSNP location, noted as After cis-eSNPs were identified, the genotype nth cis-eSNP for sample i, respectively, and N is the total number of cis-eSNPs.Given sample genotypes measured by SNP array and inferred from cis-eSNPs, the sample identity similarity between the two genotypes is defined as Similar to eSNPs, genotypes of SNPs are also associated with DNA methylation patterns and are called mSNPs i can be inferred from the associated probe methylation level i is inferred by comparing its methylation level i at the cis-mSNP location, noted as After cis-mSNPs are identified, the genotype nth cis-mSNP for sample i, respectively, and N is the total number of cis-mSNPs.Given sample genotypes measured by SNP and inferred from cis-mSNPs, the sample identify similarity between the two genotypes is defined as DNA methylation is a common epigenetic signal that regulates gene expression levels. Increased methylation at CpGs sites near gene promoter region is associated with gene repression Before aligning methylation and mRNA profiling data, we rank transformed both gene expression and methylation profiling data for each methyl probe or gene expression probe as  samples . Given ade novo mutations. Increasing the DNA copy number of a gene can increase its expression level. Instead of genotype, CNV data was aligned to gene expression and DNA methylation profiles in TCGA data sets. Cis regulation pairs and identity similarity scores were defined similarly as described above for methylation-mRNA pairs.Copy number variations (CNVs) of genome regions are commonly associated with diseases and may be inherited or occur by Multiple omics data surveying different molecular traits pertaining to the same set of samples were mapped according to the flow diagram in Match by gender. There is no ambiguity for the gender inferred from an omics profile. Any matched pair of omics profiles should have consistent gender information.Match by SNP-mRNA based identity similarity . After cis-eSNPs are identified, identity similarity vice versa as iG has the highest similarity with an unmatched mRNA profile jE among all mRNA profiles, and the unmatched mRNA profile jE has the highest similarity with iG among all genotype profiles. If there is a reciprocal best match, then the SNP and mRNA profiles are linked and sample labels are updated by comparison with mapping results based on other identity similarities.profiles . If the Match by SNP-methylation based identity similarity . After cis-mQTL are identified, the identity similarity score shown in . As abovMatch by mRNA-methylation based identity similarity . After cis methylation-mRNA probes are identified, the identity similarity score om pairs . If the Match by trio . For the samples with all three types of data available, the source of any sample label mis-matches can be identified Click here for additional data file.Figure S2The cross-aligned sample pair (TCGA-BH-A18T-01A and TCGA-BH-A18K-01A) identified by methylation-mRNA comparison was cross-aligned based on miRNA and mRNA comparison. Similarity scores based on cis miRNA-mRNA were around zero for the same labels but similarity scores for swapped pairs were the highest in both samples. Combined with the results shown in (TIF)Click here for additional data file.Figure S3PRKY was used as a gender marker for Illumina HumanMethylation27 Beadarray. For PRAD, for which only the HM450 platform is available, the methyl probe “cg04042030” corresponding to TBL1Y was used. Red, sample predicted to be female; blue, sample predicted to be male. The consistency between clinical and predicted gender is reported in Gender prediction based on methylation probe intensity in 12 cancer types in the TCGA dataset. The raw intensity of a y-chromosome probe was estimated by summation of the methylated and unmethylated channel. The methyl probe “cg20401529” corresponding to (TIF)Click here for additional data file.Figure S4Examples of mis-aligned pairs of mRNA and methylation profiles in the TCGA COAD and LUSC datasets. The similarity score for the same sample pairs based on cis methylation-mRNA pairs was not significantly higher than that of other pairs, indicating mis-alignment.(TIF)Click here for additional data file.Table S1Samples of mismatched gender information between clinical annotation and inference from multi-omics data . Red ones are mismatched with respect to clinical annotation.(XLSX)Click here for additional data file.Table S2Numbers of cis pairs in each round of alignment corresponding (XLSX)Click here for additional data file.Table S3Gender inference based on methylation probe intensity in multiple cancer data sets in TCGA. The prediction  is compa(XLSX)Click here for additional data file.Table S4Comparison of MODMatcher and MixupMapper sample alignments between SNP and mRNA profiles on the same dataset. MODMatcher was applied into 8 dataset including genotype and mRNA profiles examined by MixupMapper. MODMatcher and MixupMapper generated the same result for 6 dataset and there are small differences in for other two dataset.(XLSX)Click here for additional data file.Table S5Qualities of sample matching results based on MixupMapper and MODMatcher. For the two datasets (Choy CHB+JPT and Choy YRI) where ModMatcher and MixupMapper results were different as shown in (XLSX)Click here for additional data file.Table S6Samples pairs in the Choy CHB+JPT data set identified by MODMatcher.(XLSX)Click here for additional data file.Table S7Samples pairs in the Choy YRI data set identified by MODMatcher.(XLSX)Click here for additional data file.


Occult endometrial cancer is occasionally discovered in women with Lynch syndrome undergoing risk-reducing hysterectomy. The case presented here demonstrates that preoperative endometrial sampling can help detect these occult cancers; however, there are currently no recommendations for this preoperative intervention. A 50-year-old woman with Lynch syndrome underwent endometrial sampling prior to planned risk-reducing hysterectomy and bilateral salpingo-oophorectomy. The endometrial biopsy demonstrated a serous endometrial cancer. The patient was counselled regarding the diagnosis and revised operative plan, which now included staging, prior to surgery. Although the prevalence of occult endometrial cancer at the time of risk-reducing surgery in women with Lynch syndrome remains unknown, preoperative endometrial sampling may allow for improved patient counselling and surgical planning in this population, and can help avoid a subsequent surgery for staging. Women with Lynch syndrome have an autosomal dominant mutation in one of the DNA mismatch repair genes resulting in an increased lifetime risk for colorectal and endometrial cancer in addition to cancers of the ovary, stomach, hepatobiliary tract, pancreas, small bowel, urinary tract, and central nervous system. The current National Comprehensive Cancer Network (NCCN) guidelines recommend that individuals with Lynch syndrome undergo colorectal cancer screening with colonoscopy . There iA 50-year-old, gravida zero, Ashkenazi Jewish woman underwent screening for Lynch syndrome. Her paternal grandfather was diagnosed with colon cancer at age 80, paternal uncle diagnosed with colon cancer at age 50, and two paternal second cousins diagnosed with premenopausal endometrial cancer. Both the patient and her father were found to have the Lynch syndrome mutation MSH6 3959del4, which results in premature truncation of the MSH6 protein at amino acid position 1325 [The patient had no personal history of cancer or gynaecologic pathology and reported regular menses with a 20-year history of oral contraceptive use. The patient underwent a pelvic ultrasound which demonstrated an 8 mm endometrial echo with heterogeneity, likely representing small benign endometrial polyps. She was referred to a gynaecologic oncologist who counselled her regarding the option for risk-reducing surgery, which would include total hysterectomy and bilateral salpingo-oophorectomy. The gynaecologic oncologist performed an endometrial biopsy in the office after explaining to the patient that despite normal menses and a lack of gynaecologic symptoms, a preoperative diagnosis of endometrial cancer would affect the surgical plan and therefore a preoperative endometrial biopsy could provide valuable information. The endometrial biopsy demonstrated endometrial serous adenocarcinoma . The patThe patient underwent an uncomplicated laparoscopic total hysterectomy, bilateral salpingo-oophorectomy, pelvic and para-aortic lymph node dissection, omentectomy, and peritoneal biopsies. The final pathology demonstrated a stage IA uterine papillary serous cancer with no myometrial invasion. The patient received three cycles of chemotherapy with carboplatin and paclitaxel with no evidence of disease at the conclusion of treatment. She will continue screening for other Lynch-associated cancers.et al [Women with Lynch syndrome undergoing risk-reducing hysterectomy are at risk for having an occult endometrial cancer, however the magnitude of risk remains unknown. The estimated cumulative lifetime risk for endometrial cancer in Lynch syndrome varies by report and mutation, ranging from 21–71%. The MSH6 3959del4 mutation (as in the current case) is associated with the greatest risk of endometrial cancer, affecting 71% of women by age 70 [et al [Currently, there are no recommendations in place for endometrial sampling prior to risk reducing surgery and, in fact, gynaecologic cancer surveillance in Lynch syndrome patients remains an area of debate. Two studies have reported a lack of efficacy of screening transvaginal ultrasounds in women with Lynch syndrome , 8. Moreet al  found thet al . Women wet al . As a reet al , 2.et al [The reported cases of occult endometrial cancer discovered at the time of risk-reducing hysterectomy pose the question of whether endometrial sampling should be a routine component of the preoperative evaluation. Our case is another example of an occult endometrial cancer but additionally it highlights the clinical benefit of obtaining information on a cancer diagnosis prior to surgery. The patient described in this case was diagnosed with a serous endometrial cancer prior to surgical intervention. Patients with Lynch syndrome can have either type I (endometrioid) or type II (high-risk histologic subtypes) endometrial cancers. Broaddus et al  performeet al , 13. RisFurther prospective studies are required to determine the true prevalence of occult endometrial cancers at the time of risk-reducing surgery. However, in the meantime, surgeons may want to consider preoperative endometrial sampling in women with Lynch syndrome undergoing risk-reducing surgery, especially if in a small cohort the incidence was as high as 12.5%. As in this case, the diagnosis of cancer enabled a pre-operative discussion with the patient, and this in turn helped to avoid a second surgery for staging.There are no conflicts of interest to report.


Membrane potentials display the cellular status of non-excitable cells and mediate communication between excitable cells via action potentials. The use of genetically encoded biosensors employing fluorescent proteins allows a non-invasive biocompatible way to read out the membrane potential in cardiac myocytes and other cells of the circulation system. Although the approaches to design such biosensors date back to the time when the first fluorescent-protein based Förster Resonance Energy Transfer (FRET) sensors were constructed, it took 15 years before reliable sensors became readily available. Here, we review different developments of genetically encoded membrane potential sensors. Furthermore, it is shown how such sensors can be used in pharmacological screening applications as well as in circulation related basic biomedical research. Potentials and limitations will be discussed and perspectives of possible future developments will be provided. However, this method bears some considerable disadvantages: (i) cells need to be mechanically disturbed by the glass pipette; (ii) spatial information is limited to a cell or a patch of membrane without simultaneous recording of the potential distribution across the cell membrane; and (iii) moving cells, like cardiomyocytes within an intact beating heart can not be characterized. All these limitations can be overcome by using contact-free optical read-outs. All optical sensors for investigations of the membrane potential developed so far, independent of whether they rely on small molecule dyes or genetically encoded chromophores, are based—directly or indirectly—on membrane potential-induced changes of fluorescent properties. Fluorescence read-out has general advantages over absorption detection [The membrane potential, especially the action potential of muscles and nerves, has been measured since the middle of the 19th century ,3, usingetection . Neverthetection . This stet al. [Small molecule dyes have a number of advantageous properties, but suffer from the unspecific staining of all cell types. A possible alternative is genetic targeting using tissue specific promotors. In addition to the fluorescent protein based voltage sensors (see below), it is worthwhile to mention the combination of genetic targeting and conventional organic chromophores . In a pret al.  reportedet al. .Optical membrane potential sensors, whether small molecules, genetically encoded, or combinations thereof, share the property of reporting primarily membrane potential changes and not an absolute voltage. Ratiometric sensors allow in principle a calibration to absolute voltage, but obstacles like different bleaching of donor and acceptor in FRET based sensors render such procedures difficult. Another approach is to consider time domain based changes of photopysical properties like fluorescence lifetime imaging. In line with such considerations, a special microbial rhodopsin was engineered, where the temporal dynamics of the fluorescence was read out in pump-probe experiments reaching a voltage accuracy of 10 mV . AlthougGenetically encoded membrane potential sensors, fluorescent protein based voltage sensors or optogenetic voltage reporters are different synonyms for the same kind of membrane potential probes that are termed GEVIs  throughout this review. Beside all varieties throughout the genesis of GEVIs, they all share voltage sensing domains that are based on (or part of) an integral membrane protein, which makes GEVIs a nice example for the Special Issue “Membrane Protein Based Biosensors”.Drosophila Shaker K+-channel [+ channel Kv2.1 coupled to a CFP/YFP FRET pair in sequence and was named VSFP1 [+ channel [Ciona intestinalis Voltage Sensor-containing Phosphatase (Ci-VSP) [The first voltage sensors solely comprising genetically encoded proteins  comprised a wtGFP fused to the C-terminus of the -channel . Later, -channel . The seced VSFP1 . This seed VSFP1 . The thi channel . These t channel . In addi channel ,18,20. T(Ci-VSP) , or volt(Ci-VSP) .The Ci-VSP domain was chosen by two groups of the same institution  that independently developed what is now termed VSFP2.x  and Mermin vivo [A further line of development explored alterations in the fluorescent entities: circular permutated fluorescent proteins  and red-in vivo .Nematostella vectensis) and zebrafish (Danio rerio), also resulted in sensors with a fast kinetic response (2–5 ms) but with rather small intensity changes (0.3% per 100 mV voltage change) [All GEVIs so far share the property of a fast and a slow kinetic response component. While the fast component results from sensing currents within the voltage sensing domain, the slow component is a consequence of the voltage-dependent conformational change in the probe . The Knö change) .Gallus gallus) and zebrafish (Danio rerio) was reported to improve the temporal response, but at the expense of the response amplitude [Based on Mermaid, an improved GEVI was designed using a similar rational as for the VSFP3.x probes  but takimplitude .Gallus gallus) and displays around 29% fluorescence change per 100 mV voltage change [According to an initial report, the Accelerated Sensor of Action Potentials (ASAP1) is currently the best non-ratiometric GEVI in this group of voltage sensitive fluorescent proteins . It is be change . In addie change .A completely different concept for GEVI design is based on the use of microbial opsins  and resuThe microbial opsin-based GEVIs were improved ever since leading to new versions of Arch, like Arch-EEN and Arch-EES , Archer1Leptosphaeria maculans) to perform FRET resulted in the development of MacQ-GEVIs with a good responsiveness of around 20% per 100 mV of voltage change and an activation response of the fast component of 2.2 ± 0.2 ms representing approximately 74% of the total signal  have reached a level that allowed the transition from photometric measurements of individual cells to area detectors . The latConceptual studies of cardiotoxicity screens based on GEVI have been performed with “Mermaid”, a sensor introduced in 2008 . MermaidDanio rerio) expressing Mermaid under the control of a cardiac specific promotor [in vivo imaging of voltage dynamics in a whole heart. Although this study is eminent for having the first optical in vivo potential recordings of the entire heart, transfer from zebra fish cardiac physiology to relevant tasks of mammalian and human circulation research is rather equivocal.Furthermore, a transgenic zebrafish (promotor  demonstrDifferentiating cardiomyocytes from embryonic or induced pluripotent stem cells (iPS-cells) is becoming increasingly popular with a wide variety of applications ,69. HoweAlthough ArcLight, which was introduced in 2012 , is not etc. it would be advantageous to perform these measurements with tissue-specific targeted GEVIs. This would enable researchers to perform in situ recordings, as done for genetically encoded calcium sensors [Mapping of action potentials in excised hearts is a popular method for exploring pathophysiological processes preferentially in animal models. Electrode arrays have been used for such purpose, but they have a limited spatial resolution . An alte sensors .in vivo was reported for zebrafish using Arch(D95N) as part of a dual function calcium and voltage reporter (CaViar) [The first optical mapping of the heart with GEVIs (CaViar) . In this(CaViar) .Recently, a report based on a transgenic mice line expressing VSFP2.3 introduced the methodology to mammals . As depiThe development of GEVIs is not yet as mature as genetically encoded calcium indicators (GECIs) . Both seAlthough the development of the GEVIs was initially led by applications in neuroscience , the exaFuture sensors will further drive forward the unifying advantageous properties of single GEVIs. This will enable superior properties of GEVIs, such as a combination of high fluorescence intensity and high dynamic range, which will allow applications in non-excitable cells. Red or far-red GEVIs will allow measurements combining several sensors, e.g., phosphorylation probes  in combiWith these improvements, the general applicability of GEVIs will rise and render it a powerful extension of traditional electrophysiology. The latest progress in both GEVI development and imaging technology may bring optogenetic readouts more in line with classical current-clamp measurements and may for particular applications such as those described above even outperform them. This may especially hold true in combination with optogenetic induction of action potentials using channelrhodopsin or related proteins .


Microspore embryogenesis describes a stress-induced reprogramming of immature male plant gametophytes to develop into embryo-like structures, which can be regenerated into doubled haploid plants after whole genome reduplication. This mechanism is of high interest for both research as well as plant breeding. The objective of this study was to characterize transcriptional changes and regulatory relationships in early stages of cold stress-induced wheat microspore embryogenesis by transcriptome and small RNA sequencing using a highly responsive cultivar.in vitro anther culture: directly after induction by cold-stress treatment and microspores undergoing the first nuclear divisions. A de novo transcriptome assembly resulted in 29,388 contigs distributing to 20,224 putative transcripts of which 9,305 are not covered by public wheat cDNAs. Differentially expressed transcripts and small RNAs were identified for the stage transitions highlighting various processes as well as specific genes to be involved in microspore embryogenesis induction.Transcriptome and small RNA sequencing was performed in a staged time-course to analyze wheat microspore embryogenesis induction. The analyzed stages were freshly harvested, untreated uninucleate microspores and the two following stages from This study establishes a comprehensive functional genomics resource for wheat microspore embryogenesis induction and initial understanding of molecular mechanisms involved. A large set of putative transcripts presumably specific for microspore embryogenesis induction as well as contributing processes and specific genes were identified. The results allow for a first insight in regulatory roles of small RNAs in the reprogramming of microspores towards an embryogenic cell fate.The online version of this article (doi:10.1186/s12870-016-0782-8) contains supplementary material, which is available to authorized users. Triticum aestivum) is one of the globally most important crops that amount for 20 % of the human calorie consumption . This Transcriptome Shotgun Assembly project has been deposited at DDBJ/ENA/GenBank under the accession GDTJ00000000. The version described in this paper is the first version, GDTJ01000000.The data sets supporting the results of this article are available in the NCBI BioProject repository, under accession PRJNA297977, [


Since many decades, it is known that viral infection can lead to a strong immune activation. This partially explains the strong association of viral infection with autoimmunity, immunopathology during chronic virus infections and complications during transplantation. One explanation for the strong immune stimulatory effects of viruses is the expression of specific patterns . Pathogen-specific patterns can be recognized by endogenous receptors (e.g.: Toll -like receptors), which are known to induce a strong innate immune activation. Whether further immunological mechanisms contribute to virus-specific immune activation is current topic of research.+ macrophages and conventional dendritic cells) selectively allowed replication of viruses. This enforced viral replication led to an exorbitant propagation of viral antigens and viral RNA. Viral antigen led to massive activation of the adaptive immune system, whilst viral RNA activated innate immunity. Therefore the mechanism of enforced viral replication partially explains the association of strong immune activation with virus infection.In the recently published work ,2, we id+ macrophages express the protein UBP43 (gene name: Usp18). UBP43 is a cellular interferon type I (IFN-I) inhibitor. Therefore, due to expression of UBP43, IFN-I could not induce an antiviral status in CD169+ macrophages. Thus VSV, once it infected CD169+ macrophages, showed strong replication in this particular cell type. Other types of macrophages (Kupffer cells and red pulp macrophages) did not show VSV replication after infection. The enhanced replication of VSV in CD169+ macrophages induced a strong immune activation. Genetic depletion of Usp18 or depletion of CD169+ macrophages limited enforced viral replication and resulted in a reduced activation of the adaptive and innate immune response. As a consequence virus could not be controlled resulting in fast death after infection. Together our data show that replication of VSV in specific splenic niches is essential for innate and adaptive immune activation and for surviving an infection with cytopathic VSV.First, we analyzed the role of enforced viral replication during infection with cytopathic vesicular stomatitis virus (VSV). We demonstrated that CD169+ T cells, which usually develop during LCMV infection, show cross-reactivity to pancreatic beta cells of RIP-GP mice. Therefore RIP-GP mice develop diabetes during infection with LCMV. We found that induction of LCMV-specific CD8+ T cells as well as LCMV induced innate immune response depended on enforced viral replication. Genetic knockdown of Usp18, depletion of CD169+ macrophages and conventional dendritic cells or pharmacological inhibition of viral life cycle, blunted viral replication in the spleen and limited innate and adaptive immune activation against LCMV. Thereby the development of autoimmune diabetes was prevented.To investigate the contribution of enforced viral replication to autoimmune disorders we tested its role in the induction of autoimmune diabetes using the RIP-GP mouse model. RIP-GP mice express the glycoprotein of the lymphocytic choriomeningitis virus (LCMV) under the rat insulin promoter. LCMV-specific CD8In summary, professional innate immune cells purposely allow virus replication due to over-expression of UBP43. UBP43 expression and accelerated virus replication was essential to activate the antiviral innate and adaptive immune response. At the one hand this mechanism is absolutely required to protect from fatal virus infection, at the other hand it also strongly contributes to induction of virus induced autoimmune diabetes. In the future, it will be investigated in which other diseases this mechanism could play a role, and whether it may also have clinical implications.