Lactuca sativa L.) seed germination. Different lettuce genotypes respond differently to higher temperatures or thermal stress. In this study, we evaluated the germination performance of 304 lettuce accessions incubated at three temperature settings, 21 °C, 28 °C and 35 °C, respectively, for 40 h. At 21 °C, seeds of all 304 accessions germinated with very well an average germination percentage of 87.72%; at 28 °C, the average germination percentage dropped to 42.84% and at 35 °C, the germination decreased to 1.01%. Then, we investigated changes in metabolic profiles of lettuce seed response to thermal stress using an untargeted metabolomics approach. Results suggested that seeds of thermal-sensitive and thermal-tolerant cultivars employed different metabolic strategies in response to thermal stress during germination. Thermal-sensitive buds accumulated more significant amounts of organic acids, amino acids, sugars, sterols, phenolic compounds and terpenoids compared to thermal-tolerant buds at 21 °C. Thermal-tolerant lettuce cultivar accumulated higher concentrations of amino acids, organic acids, sugars, sesquiterpene lactones, sterols, and fatty acids derivatives during the germination at 35 °C compared to germinated at 21 °C. This investigation paves the way to link the metabolomics to other external and internal factors affecting lettuce seed germination under thermal stress.Temperature strongly influences lettuce ( Thermoinhibition refers to the phenomenon in which viable seeds failed to germinate at high temperature and it is recognized as an adaptive regeneration strategy for the cool-season crop to prevent seeds from germination at inappropriate high-temperature particularly in summer that may hamper the growth and development of the young plant . In agriDue to its significance in both basic study and applied crop production, this fascinating phenomenon of thermoinhibition has been investigated for a long time. In the 1960s, researchers found that external factors such as light  and inteMetabolomics has been widely employed to establish the connections between genotype and phenotype and is capable of identification and quantification of different classes of metabolites simultaneously and unbiasedly ,12. Non-In this study, we evaluated the germination percentage of 274 lettuce accessions from Shanghai Agricultural Gene Center (SAGC) and 30 commercial cultivars purchased from the local market after being incubated for 40 h at 21 °C, 28 °C and 35 °C, respectively, in a germination chamber. Based on the obtained results, two lettuce accessions, one thermotolerant entry  and one thermosensitive entry  were selected for further study using a metabolomics approach. We combined gas chromatography-mass spectrometry (GC/MS) and ultra-performance liquid chromatography-ion mobility spectrometry quadrupole time-of-flight mass spectrometry (UPLC-IMS-QTOF/MS) platforms to reveal the primary and secondary metabolites among the seed samples of the two selected accessions of dry seeds (seeds without germination tests), germinated seeds and ungerminated seeds at two temperature settings, 21 °C and 35 °C, respectively. We anticipate that this investigation will detect differences among the seed samples of different genotypes under different treatments and gain insight regarding the metabolic mechanism of lettuce seed thermoinhibition.The 304 lettuce accessions used in this study were collected from the 34 countries . A largep < 0.01), indicating lettuce seeds are sensitive to high-temperature stress  or 35 °C . In addition, there were no relationships between 100-grain weight and seed color  or 100-grain weight and seed source . However, there was a negative relationship between relative percent germination and 100-grain weight at 28 °C , but not at 35 °C.There was no relationship between dark and white seed coats on germination rates at 28 °C  and a thermotolerant variety s15k0106), were selected to investigate the metabolic mechanisms of seed germination at high temperature. A non-targeted metabolomics strategy combining GC/MS and UPLC-IMS-QTOF-MS was used to simultaneously detect and enable relative quantification of primary and secondary metabolites of lettuce [k0106, we2(cum) value of 0.491 was established. PC1 and PC2 explained 53.6% of the total variation, and clear separation was observed among the six different groups; the optimal germination temperature treatments (C106 and C62) were distributed far from the center of the coordinate axis , and variable importance in projection (VIP) score (> 1.0) of the metabolites in each pair of comparisons and then identified these differential metabolites using online databases and our in-house database.We performed unsupervised PCA analysis to determine the variations in metabolites between different cultivars and at different germination temperatures. A five-principle component-based robustness PCA model with a Qate axis . To idenl-methionine, l-ornithine, l-asparagine, l-methionine), as well as arabinitol and caffeoyl-hexose isomer 2  and thermosensitive seeds (N62); 11 of these metabolites were putatively annotated . N106 seisomer 2 .l-threonic acid, 4-hydroxybutanoic acid, malic acid), amino acids , sugars (glucose and fructose), oleic acid, (Z)-, sterols , terpenoids  and polyphenols  than C106  and thermosensitive seeds (C62) that were germinated at 21 °C, which led to the selection of 2175 candidate metabolites of which 39 were putatively identified . C106 hahan C106 . Overalll-isoleucine, l-5-oxoproline, l-threonine, l-alanine, l-glutamic acid, l-valine, l-aspartic acid, l-proline, l-ornithine, l-methionine, l-leucine), sesquiterpene lactones (lactucopicrin), carbohydrates, including fructose, xylitol, arabinose, 1-(sn-Glycero-3-phospho)-1D-myo-inositol and lipid derivatives -, palmitic acid, stearic acid, arachidic acid and 2-linoleoylglycerol) in s15k0106 seeds germinated at 35 °C (HY106). However, only some carbohydrates, including meso-erythritol and arabinitol, were observed to be lower in seeds germinated at 35 °C (HY106) than buds of the same cultivar at germinated at 21 °C , dihydrocaffeic acid hexose and 15-deoxylactucin-8-sulfate in germinated buds than the non-germinated seeds (We assessed the metabolic differences between non-germinated seeds and germinated buds of the thermotolerant cultivar after incubation at 35 °C for 40 h (H106 vs. HY106). A total of 1220 differential metabolites candidates were detected, of which 39 candidates were putatively annotated . The noned seeds .Lettuce seed germination is dramatically inhibited at high temperatures and will induce thermodormancy; the resulting poor seed germination and thermodormancy have detrimental effects on lettuce production in the field . In this2 = −0.397, p < 0.01), but not at 35 °C, suggesting that seed weight might be a useful predictor of germination at 28 °C. Further germination experiments at 21 °C to 35 °C are therefore required to explore higher correlation coefficients. There are several different opinions about the center of origin of cultivated lettuce. According to Lindqvist, it probably originated from Egypt [Seed color had been previously reported to be associated with seed germination, dark-seeded lettuce is believed to have a higher percent of germination and increased seed vigor as well as lower pathogen susceptibility than white-seeded lettuce . In thisom Egypt . Accordiom Egypt ,30. In tSeed germination is a complex physiological and metabolic process that involves different classes of metabolites, including amino acids, organic acids, available sugars, antioxidants and lipids ,32,33. Il-alanine, proline, l-5-oxoproline, l-glutamic acid, and l-aspartic acid and l-ornithine) and sugars  function as compatible solute properties in Arabidopsis in response to temperature stress [2O2-producing genes  in the plant under stress [Combined using GC/MS and UPLC-IMS-QTOF/MS platforms, we profiled the primary and secondary metabolites between the seed samples of thermotolerant lettuce cultivar of germinated at appropriate temperature (21 °C) and heat temperature (35 °C). We observed that thermotolerant lettuce cultivar accumulated higher concentrations of amino acids, organic acids, sugars, sesquiterpene lactones, sterols and fatty acids derivatives during the germination at 35 °C compared to germinated at 21 °C. Amino acids and some carbohydrates are recognized as osmotic substances; the higher accumulation in seeds under thermal stress may contribute to the stabilization of membranes and cellular osmotic pressure . In addie stress . Aromatir stress . Brancher stress . Additior stress .Our results showed that pyruvic acid and TCA intermediates, such as butanedioic acid, fumaric acid, malic acid and citric acid, had higher accumulation in HY106 compared to C106. Pyruvic acid plays an essential precursor for the TCA cycle, and its accumulation results in the increased levels of TCA intermediates. In addition, the previous study reported that the activation of 4-aminobutanoic acid shunt resulted in TCA intermediates accumulation in Arabidopsis in response to dehydration due to the interplay between the 4-aminobutanoic acid shunt and the TCA cycle through various bypasses .Festuca Trachyphylla): ethyl sterols  are positively associated with heat tolerance [Notably, we observed that N106 seeds contain higher concentrations of the unsaturated long-chain fatty acids and saturated long-chain fatty acids than those in seeds of the thermosensitive cultivar. These results imply that heat-resistant of lettuce seed germination might be associated with lipids metabolism. Lipids are energy sources in seeds during embryo development, and the accumulation of high abundances of fatty acids is associated with energy metabolism in seeds . Moreoveolerance . Fatty aolerance . To mainolerance .Interestingly, we observed significantly higher accumulation of organic acids, amino acids, sesquiterpene lactones, lipids and carbohydrates in the buds of the thermotolerant accession s15k0106 when germinated at 35 °C (HY106) than when germinated at 21 °C (C106). A similar metabolic strategy was detected between s13k0062 buds germinated at 21 °C (C62) and s15k0106 buds germinated at 21 °C (C106). Twenty-three differential metabolites were common to both of these comparisons (HY106 vs. C106 and C106 vs. C62), suggesting that the thermotolerant cultivar s15k0106 may employ a similar metabolic strategy when germinated at high temperatures (35 °C) as the thermosensitive cultivar s13k0062 when germinated at 21 °C. While the thermosensitive s13k0062 seeds accumulated high levels of antioxidants  during germination, and the accumulation of phenolic compounds were observed to be lower in thermotolerant s15k0106 seeds during germination at 35 °C compared to 21 °C. The reason could be that the thermotolerance of lettuce seeds undergoing germination is associated with the levels of reactive oxygen species in the seed. The levels of activated oxygen species were higher in the thermosensitive cultivar than the thermotolerant cultivar, which would lead to poorer heat resistance in the thermosensitive cultivar. Collectively, thermosensitive and thermotolerant cultivars employ distinctive metabolic strategies in response to heat stress. This work identified a large number of potential metabolic targets for further in-depth investigation of acquired seed tolerance to temperature stress. Moreover, these differential metabolites could be potential biomarkers for further screening of thermotolerant lettuce varieties for breeding projects.Used in the current study were 304 lettuce accessions, of which 257 were kindly offered by the Western Region Plant Introduction Station of the US Department of Agriculture, Pullman, WA, USA; 17 were provided by World Vegetable Centre, Taiwan, China; 30 commercial cultivars were purchased from the local market in Shanghai, China. Seeds were stored at −20 °C until starting the germination test.For the germination test, three replicates of 100 seeds per accession or cultivar were placed in petri dishes (120 × 120 mm) on a single layer of Whatman #1 filter paper. A thin sponge was placed under the filter paper to retain moisture, and 4.5 mL of deionized water was added. The petri dishes were covered with lids to prevent evaporation then placed in incubators at 21 °C (proper temperature for lettuce seeds germination), 28 °C (upper limit temperature for lettuce seed) or 35 °C (upper limit temperature for thermotolerant cultivar seed) for 40 h in the dark to mimic the conditions of the soil at germination. Seed germination was recorded as the emergence of a radicle after incubation. Analysis of variance was performed on the germination data.Two accessions, ‘s13k0062′ and ‘s15k0106′, were selected for metabolic profiling based on their germination performance under different temperatures. At 21 °C, both ‘s13k0062′ and ‘s15k0106′ germinated well with the germination percentages of 92.67% and 76.0%, respectively. However, at 28 °C, only less than 2% of the seeds of ‘s13k0062′ germinated, while the germination percentage of ‘s15k0106′ remained at 74.67%, almost the same as that at 21 °C. Still, at 35 °C, ‘s13k0062′ had 0% germination, while ‘s15k0106′ had 39.67% germination, the highest among all 304 accessions evaluated . Thus, wFour replicates of 100 seeds per cultivar were placed in an incubator to mimic seed germination using the method described above at 21 °C and 35 °C for 40 h. After incubation, all samples were collected and stored at −80 °C for metabolite extraction.−1 L-2-chlorophenylalanine) was added. After that, samples were centrifuged at 15,000× g for 10 min, and 300 μL supernatant of each sample was transferred to a new vial and vacuum-dried at room temperature. For derivatization, 80 μL of 20 mg·mL−1 methoxyamine hydrochloride (dissolved in pyridine) was added to the vial, and samples were then transferred into an oven and incubated at 37 °C for 1.5 h. Then, 80 μL of N, O-Bis(trimethylsilyl)trifluoroacetamide solution with 1% trimethylchlorosilane was added and followed by incubation at 70 °C for 1h. Supernatants of all samples were mixed, vacuum-dried and derivatized in the same manner to prepare QC samples. 1 μL derived extract of each sample was analyzed by using an Agilent 7890A-5975C GC/MS system  coupled with a DB-5MS capillary column (30 m × 0.25 mm × 0.25 μm). The GC conditions were set as inlet temperature, 280 °C; After 6.5 min of solvent delay, the initial GC oven temperature was 60 °C; 1 min after injection, the GC oven temperature was raised to 300 °C at 5 °C min−1, and maintained at 300 °C for 11 min; transfer line temperature, 280 °C; ion source temperature, 230 °C; carrier gas (He) flow rate, 1mL min−1. MS condition: ion source, electron ionization; ionization energy, 70 eV; scan mode, full scan; scan range, m/z 33–600. The mixture of Supelco C7–C40 saturated alkanes standard ran prior to the samples to calculate the retention index of each feature. The QC samples were run at the start, middle, and end of the analysis.A recently published extraction protocol  was follv/v). Samples were then sonicated at 25 °C for 30 min and kept in the refrigerator (4 °C) for 12 h, followed by centrifugation at 12000× g for 10 min. 500 μL of the supernatant was collected for further analysis. QC samples were prepared by mixing the supernatant of all samples.The sample for UPLC-MS analysis was prepared and analyzed, as described previously . In brie−1; UPLC conditions: gradient elution, 0–3 min, 0% B; 3–3.1 min, 0–5% B; 3.1–6 min, 5 to 20% B; 6–11 min, 20–50% B; 11–15 min 50 to 100% B, 15–17 min, 100% B, then initial conditions were restored for 5 min to equilibrate the column. For MS conditions, ion mode, negative; capillary voltage, 1.5 kV; source temperature, 115 °C; desolvation temperature, 500 °C; desolvation gas flow, 1000 L h−1; scan range, m/z 50–1000; collision energy, 20–40 eV. Data acquisition mode was performed in HDMSE (data-independent acquisition type in ion-mobility). A 5 μL of 0.2 mg mL−1 EGCG was added as an internal standard. QC samples were inserted into each sample branch.Samples (3 μL) were then analyzed by an Acquity class UPLC coupled with Vion IMS QTOF MS  using an Acquity UPLC HSS T3 column . Mobile phase: Water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B); flow rate: 0.4 mL minm/z,), sample information , relative abundance  was prepared and submitted to MetaboAnalyst . After that, the data matrix was performed three categories of normalization, including normalization by median, log transformation and auto-scaling via online data analysis software MetaboAnalyst embedded algorithm.Raw data acquired from GC/MS and UPLC-IMS-QTOF/MS were pre-processed by LECO Chroma TOF and Waters Progenesis QI, respectively. The raw data was performed in the pre-process procedures, including peak picking, baselining and alignment. Then, the pre-processing data matrix containing feature name ; 2. the germinated seeds of the two accessions at 21 °C (C106 vs. C62); 3. the germinated seeds of ‘s15k0106′ at 21 °C and 35 °C (HY106 vs. C106); and 4) the seeds of ‘s15k0106′ germinated and non-germinated at 35 °C (HY106 vs. H106). The study design is shown in The principal component analysis (PCA) of all samples and QCs was performed in SIMCA-P software . To identify potential variables that contribute to differentiation of four pairs of comparison, fold change and student’s t-test value were calculated, and VIP value was calculated by conducting the partial least squares-discriminant analysis (PLS-DA). All these parameters were obtained by the embedded algorithm in MetaboAnalyst.p-value < 0.01. Then, these ‘candidates’ were marked for compound identification to explore their biological roles during seed germination.The differential metabolites were screened by parameters including fold change > 2, VIP value > 1, and 2 fragments. All matching results were then supervised manually.Candidates from GC-MS were identified through compared with reference and online database including our in-house lettuce metabolites library , NIST 20In conclusion, the germination of lettuce germplasm reduces as temperature increases. Artificial selection has improved the germination rates of lettuce under heat stress. Our comparative metabolomic analysis revealed that the accumulation of organic acids, amino acids, terpenoids, phenolic compounds, carbohydrates and lipids are involved in lettuce seed germination and thermoinhibition. Thermosensitive seeds accumulated more significant amounts of organic acids, amino acids, sugars, sterols, phenolic compounds and terpenoids compared to thermotolerant seeds at 21 °C. Thermotolerant lettuce cultivar accumulated higher concentrations of amino acids, organic acids, sugars, sesquiterpene lactones, sterols and fatty acids derivatives during the germination at 35 °C compared to germinated at 21 °C. Overall, our findings indicate the complexity of lettuce germination under abiotic stress conditions. Seeds of thermal-sensitive and thermal-tolerant cultivars employed different metabolic strategies in response to thermal stress during germination. Moreover, heat-resistant accessions were identified for future breeding programs that aim to develop new cultivars suitable for extended summer production in temperate or tropical climates when soil temperatures are higher than the optimal germination temperature. Further studies are now required to assess the ability of these accessions to germinate rapidly at higher temperatures under field conditions.


TM Biotechnology). Cells were characterized by immunophenotype and multilineage differentiation potential. Upon an initial cell adhesion in the spinner flask of 35 ± 2.5%, culture reached a maximal cell density of 2.6 ± 0.1 × 105 at day 7, obtaining a 15 ± 1-fold increase. The implementation of the culture in the 500-mL mini-bioreactor presented an initial cell adhesion of 22 ± 5%, but it reached maximal cell density of 2.7 ± 0.4 × 105 at day 7, obtaining a 27 ± 8-fold increase. Importantly, in both stirred systems, cells retained their immunophenotype and multilineage differentiation potential . Overall, the scalability of this microcarrier-based system presented herein is of major importance for the purpose of achieving clinically meaningful cell numbers.Mesenchymal stem/stromal cells (MSC) are promising candidates for cell-based therapies and for the promotion of tissue repair, hence the increase of clinical trials in a worldwide scale. In particular, adipose tissue-derived stem/stromal cells (AT MSC) present easy accessibility and a rather straightforward process of isolation, providing a clear advantage over other sources. The high demand of cell doses (millions of cells/kg), needed for infusion in clinical settings, requires a scalable and efficient manufacturing of AT MSC under xenogeneic(xeno)-free culture conditions. Here we describe the successful use of human AB serum [10%(v/v)] as a culture supplement, as well as coating substrate for the expansion of these cells in microcarriers using (i) a spinner flask and (ii) a 500-mL mini-bioreactor (Applikon Mesenchymal stem/stromal cells (MSC), characterized as multipotent cells, have been the focus of academia, as well as cell therapy industries, due to their ability to differentiate into a variety of lineages , as well as their paracrine activity which modulates inflammation and other cell processes . Becausein vitro  , MSC are considered a heterogeneous cell population characterized by spontaneous adherence to plastic, positivity for CD105, CD73, and CD90 and negativity for the expression of CD45, CD34, CD14 or CD11b, CD79 or CD19, and human leukocyte antigen class II, and ability to differentiate oblasts) . MSC canoblasts) . Neverthoblasts) , divergioblasts) .5 cells per gram of tissue) compared to other sources (Limitations related to the isolation procedure include low percentage of the target cells in the collected biopsy and a highly invasive collection method (BM), high number of contaminants (AT) and low yield of number of cells per unit (UCM) . In part sources  and are  sources . AT MSC  sources . Moreove sources .ex vivo expansion of MSC involves 2D static culture systems, typically employing fetal bovine serum (FBS) for culture medium supplementation, some efforts have been made to substitute this supplement due to the drawbacks intrinsic to the serum .Adipose tissue samples (500 mL) from liposuction  were provided by the Clinical Hospital , after ethical consent (Process 375415/2013). Samples were washed with Phosphate Buffered Saline Solution (PBS) and homogenized. After resting, two distinct phases were formed, an AT supernatant and an aqueous infranatant phase, the latter being discarded. After the wash, the sample was incubated with 0.1% Collagenase type II  at 37°C for 30 min. Furthermore, the sample was incubated with Ammonium-Chloride-Potassium solution  for 10 min to lyse red blood cells, diminishing the presence of blood contaminants. Subsequently, the highly viscous solution was subjected to a filtration step and centrifuged. The supernatant was discarded and the pellet was resuspended in alpha Minimum Essential Eagle Medium (a-MEM)  supplemented with 10% (v/v) Human AB serum (AB HS) [in-house produced ] and 1% 2 in a-MEM supplemented with 10% (v/v) AB HS. After reaching 80–90% confluence, cells were detached using TrypLETM 1x  and subcultured at the same cell density until passage 3 or 4 (P3/P4). T-flasks were maintained at 37°C under a 5% CO2 humidified atmosphere, with medium exchange every 3 to 4 days. The Trypan Blue (0.4%) exclusion method was used to determine cell number and viability.Upon thawing, cryopreserved cells were seeded (passages 4–5) on T-flasks at a density of 3000 cells/cm6 cells were seeded, making a total final volume of 40 mL. For the first 24 h, a continuous stirring regimen was set at 30 rpm, and from this point onward, agitation was set at 40 rpm. At day 2 of culture, 40 mL of fresh culture medium was added to the spinner flasks, and from thereon, 25% (v/v) of culture medium was changed every day.For the dynamic culture of AT MSC, 100 mL spinner flasks  equipped with 90° paddles and a magnetic stir bar were used, with a working volume of 80 mL. Plastic microcarriers  were coated by incubating with a-MEM + 10% (v/v) AB HS overnight. After adding 20 g/L of the pre-coated plastic microcarriers to the spinner flasks, 4 × 10n = 3).Cell counting was performed by removing a microcarrier-cell suspension sample of 0.5 mL from the homogeneous culture. Microcarriers were then washed with 2 mL of PBS and 1 mL of TrypLE Express 1x was added. The microcarrier suspension was then incubated at 37°C for 7 min at 650 rpm using Thermomixer . Subsequently, 4 mL of a-MEM + 10% (v/v) AB HS was added to stop enzymatic activity and the cell/microcarrier suspension was filtered using a 100 mm Cell Strainer . Cell number and viability were determined using the Trypan Blue exclusion method. Cell adhesion efficiency is calculated as the percentage of cells that successfully attached to the microcarrier beads after 24 h of culture (relative to day 0). MSC from three independent donors were used (TM Biotechnology) equipped with a three-blade pitched impeller was used. The culture parameters were set to: pH 7.3, 20% of DO by headspace aeration  and temperature 37°C. Pre-coating of plastic microcarriers was performed as previously described (see section “AT MSC Expansion in Spinner Flasks”). Three bioreactor experiments with independent cell donors were performed (n = 3). For each run, 12.5 × 106 cells were inoculated with 20 g/L of pre-coated plastic microcarriers in a final volume of 150 mL. At day 3 of culture, 100 mL of fresh culture medium was added to the bioreactor, and from thereon 25% (v/v) of culture medium was changed every day. Agitation was set at 85 rpm until day 2, 95 rpm until day 5 and 105 rpm from thereon after. The increase in agitation was an empirical parameterization to counterwork the increasing settling rate of the microcarriers due to a gradually higher occupancy by the cells, according to Stokes’ law . The analyzes are based on the specificity of immobilized enzyme electrodes for a single target analyte, allowing a rapid, accurate and largely interference free quantification. The range of measurement of glucose and lactate is 0.05–25 g/L and 0.05–2.70 g/L, respectively.At the beginning and at the end of bioreactor culture, cells were analyzed by flow cytometry  using a panel of mouse anti-human monoclonal antibodies against CD14 (PE-conjugated), CD19 (APC-conjugated), CD31 (PE-conjugated), CD34 (PE-conjugated), CD45 [fluorescein isothiocyanate (FITC)-conjugated], CD54 (PE-conjugated), CD73 (PE-conjugated), CD80 (PE-conjugated), CD90 (PE-conjugated), CD105 (PerCP-conjugated), and HLA-DR (PerCP-conjugated) . A minimum of 10 000 events were collected for each sample and the data was acquired using FACSCalibur.2 on a 24-well plate  with culture medium. At confluence, cells were induced to differentiate into osteocytes and adipocytes using StemProTM Osteogenesis and Adipogenesis Differentiation Kit , respectively. Culture medium was exchanged twice a week. Adipogenesis was observed after 14 days for lipid droplets (Sudan II-Scarlet) and osteogenesis after 21 days for mineralized bone matrix deposition (von Kossa). For chondrogenic differentiation, 1 × 107 cells were plated in droplets (5 μL) on Ultra-Low attachment multi- well plates . The plate was left in the incubator for 30 min and afterward StemProTM Chondrogenesis Differentiation medium  was added. Total medium exchange was performed two times a week for 15 days. Cells were then stained with Alcian blue  for 2 h to assess proteoglycan synthesis . A one-way analysis of variance (ANOVA) was used to compare data between different groups. Tukey’s post hoc tests were carried out to determine the differences between groups.Results are presented as mean ± standard deviation (SD) of the values obtained for the different cell donors. The expansion of AT MSC in spinner flasks and stirred-tank bioreactor was performed using cells from three independent donors (5 (day 6), 2.3 × 105 (day 6) and 2.2 × 105 cells/mL (day 7), respectively. Although the culture using a fibronectin coating appears to present a slightly higher cell adhesion efficiency, it reached lower cell densities and presented a higher lag phase. Moreover, it took an extra day to reach similar maximum cell densities compared to any of the AB HS coatings. Considering these preliminary results, 10% (v/v) of AB HS was chosen for microcarrier coating for the further experiments.Previous work from our group demonstrated the low adhesion efficiency of AT MSC in microcarrier based-stirred systems when combined with commercially available serum-/xeno-free culture medium formulations . Therefon = 3, three independent donors) were cultured in spinner flasks using the microcarrier coating of 10% (v/v) of AB HS previously established. After 24 h of continuous stirring, a cell adhesion efficiency of 35 ± 2.5% was achieved, attaining a maximal cell density of 2.6 ± 0.1 × 105 cells/mL at day 7, which represents a fold increase of 15 ± 1 , representing a fold increase of 27 ± 8  was lower than the one obtained for spinner flasks . Althougf 27 ± 8 . Table 1Glucose and lactate concentrations were measured every day throughout the culture . As can in vitro along the osteogenic, chondrogenic and adipogenic lineages. Results show that the AT MSC were positive for several markers . After cultivation, cells were enzymatically harvested and characterized regarding the immunophenotypic profile and the capacity to differentiate ® Synthemax II microcarriers (® MSC SFM XenoFree) (5 cells/mL) achieved were significantly inferior when compared to BM MSC expanded under the same conditions (95% and 3.6 × 105 cells/mL), highlighting the need to improve the expansion process of this specific cell source under xenogeneic(xeno)-free culture conditions  , the celnditions . Furthernditions . This prBased on our previous reports regarding AB HS use as culture medium supplement for MSC expansion, with cells showing similar doubling times and population doublings compared to cell cultivated with 10% (v/v) FBS supplemented culture medium , we sele® MSC SFM Xeno-Free. Despite the hindrance of not improving initial cellular adhesion, the maximal cell density achieved was significantly higher, 2.6 × 105 ± 0.1 × 105 when compared to 1.9 × 105 cells/mL obtained in previous studies (After selecting a-MEM supplemented with 10% (v/v) AB HS as the best strategy for microcarrier coating, the adhesion efficiency calculated (35 ± 2.5%) was not higher than the one determined by Carmelo et al. (2015) (36 ± 5.2%) when using StemPro studies . These r5 ± 0.4 × 105), resulting in a higher fold increase of 27 ± 8. We believe that improving cell adhesion would further increase maximal cell density. Not only cell-to-bead ratio is an important variable that affects cell adhesion, but also the microcarrier density plays an important role, as it promotes more cell-bead contact, hence improving adhesion. After initial microcarrier concentration, initial cell seeding density might be the next step for improving cell expansion in this platform. Moreover, one advantage of microcarrier-based culture systems is that by simply adding fresh microcarriers to the culture at a specific time point, we could overcome the surface area limitation  AB HS, in the Applikon mini-bioreactor. With a tighter control over pH, dissolved oxygen, agitation and temperature, we aimed to improve even further the expansion of AT MSC under stirred conditions. Despite the observed lower adhesion efficiency (22 ± 5%) compared to the spinner flask (35 ± 2.5%), we maintained the maximal cell density attained (2.7 × 10mitation , withoutAnother key feature that must be addressed is the optimization of the feeding regimen. In the present work, 25% of medium exchange was performed at day 3 onward in order to replenish nutrients and dilute toxic metabolites . When adIn vitro differentiation capacity of cells is one of the criteria proposed by ISCT to attest MSC identity after ex vivo culture. It is well known that MSC exhibit distinct differentiation levels according to their microenvironmental niche and culture conditions (nditions . In our In conclusion, we report the establishment of a platform for AT MSC expansion under xeno-free conditions using AB HS supplement in a microcarrier-based culture system, achieving a 29-fold expansion after 7 days of culture. Although The datasets generated for this study are available on request to the corresponding author.FM carried out the experiments of AT MSC expansion and wrote the manuscript. AM helped to design, develop the experiments, and wrote the manuscript. LS and FM performed the AT MSC isolation from adipose tissue samples. JC, CS, DC, and KS supervised the project. KS helped to design the experiments. All authors discussed the results and contributed to the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Atrioventricular nodal reentry tachycardia (AVNRT) is the most common manifestation of paroxysmal supraventricular tachycardia (PSVT). Increasing data have indicated familial clustering and participation of genetic factors in AVNRT, and no pathogenic genes related to AVNRT have been reported.Whole‐exome sequencing (WES) was performed in 82 patients with AVNRT and 100 controls. Reference genes, genome‐wide association analysis, gene‐based collapsing, and pathway enrichment analysis were performed. A protein‐protein interaction (PPI) network was then established; WES database in the UK Biobank and one only genetic study of AVNRT in Denmark were used for external data validation.RYR2, NOS1, SCN1A, CFTR, EPHB4, ROBO1, PRKAG2, MMP2, ASPH, and ABCC8. From the UK Biobank database, 18 genes from candidate genes including SCN1A, PRKAG2, NOS1, and CFTR had rare variants in arrhythmias, and the rare variants in PIK3CB, GAD2, and HIP1R were in patients with PSVT. Moreover, one rare variant of RYR2  in our study was also detected in the Danish study. Considering the gene functional roles and external data validation, the most likely candidate genes were SCN1A, PRKAG2, RYR2, CFTR, NOS1, PIK3CB, GAD2, and HIP1R.Among 95 reference genes, 126 rare variants in 48 genes were identified in the cases . Gene‐based collapsing analysis and pathway enrichment analysis revealed six functional pathways related to AVNRT as with neuronal system/neurotransmitter release cycles and ion channel/cardiac conduction among the top 30 enriched pathways, and then 36 candidate pathogenic genes were selected. By combining with PPI analysis, 10 candidate genes were identified, including SCN1A, PRKAG2, RYR2, CFTR, NOS1, PIK3CB, GAD2, and HIP1R, and the pathways mediated by these genes, including neuronal system/neurotransmitter release cycles or ion channels/cardiac conduction, might be involved in AVNRT.The preliminary results first revealed potential candidate genes such as  However, factors such as lack of large multiplex families, locus heterogeneity, and incomplete penetrance have hampered such efforts to identify pathogenic genes in many diseases. Recent advances in gene‐based collapsing analysis might overcome some of these limitations.To our knowledge, there are no published studies identifying the pathogenic genes in AVNRT. In the current pilot study, we examined AVNRT using whole‐exome sequencing (WES) to verify possible pathogenic genes by gene‐base burden, pathway enrichment, and protein‐protein interaction (PPI) analyses.2The study participators were identified among patients treated with radiofrequency catheter ablation at the Department of Cardiology of the Sichuan Academy of Medical Sciences and the Sichuan Provincial People's Hospital in the period from 2014 to 2017. A total of 100 unrelated ethnically matched healthy control subjects were enlisted from the visitors to the Health Evaluation and Promotion Center of the Sichuan Academy of Medical Sciences and the Sichuan Provincial People's Hospital. Upon inclusion, blood sample tests, 12 lead electrocardiograms, echocardiography, and cardiac history were recorded. The control subjects were free of any cardiovascular diseases, arrhythmia, chronic anemia, diabetes mellitus, thyroid disorders, electrolyte disturbance, systemic immune diseases, malignant tumors, or any other diseases known to cause arrhythmias. A written informed consent for genetic screening was obtained from all participants. Ethical approval for this study was acquired from the ethics committee of the Sichuan Academy of Medical Sciences and the Sichuan Provincial People's Hospital.2.1Baseline intracardiac electrophysiological studies included atrial stimulation (burst or extra stimulus pacing) and ventricular stimulation in cases. AVNRT diagnosis was set up according to published criteria and pacing maneuvers as applicable.2.2http://bio-bwa.sourceforge.net/). SAMtools , Picard (http://picard.sourceforge.net/), and GATK (http://www.broadinstitute.org/gsa/wiki/index.php/Home_Page) were used for removing duplicated reads, realignment, and recalibration. Potential single nucleotide variants (SNVs) and small insertions and deletions (indel) were called and filtered by using GATK3.7. Then, high‐confidence SNV and indel variants were noted using snpEff . Furthermore, all variants were annotated according to the control population of the 1000 Genomes Project , ExAC (http://exac.broadinstitute.org), EVS (http://evs.gs.washington.edu/EVS), the disease databases of ClinVar (http://www.ncbi.nlm.nih.gov/clinvar), and OMIM (http://www.omim.org).DNA samples were extracted from peripheral blood using the QIAamp DNA Blood Mini and Maxi Kits  according to the manufacturer's instructions. Entire exon sequences were enriched by using a SureSelect Human All Exon kit V6 , and the libraries were sequenced on the Illumina HiSeq NovaSeq platform . The average read depth was 123, and on average 96.4, 98.6, and 99.4% of exons were covered by at least 20 reads, 10 reads, and 4 reads, respectively . Qualified sequence reads were arrayed to the human reference genome (NCBI GRCh37) using the Burrows‐Wheeler Aligner. Finally, we used a genome‐wide threshold for significance of P < 1 × 10−6. A quantile‐quantile (Q‐Q) plot was used to evaluate the resulting P values.We used GATK v3.7 CombineGVCFs to combine the WES dataset with ethnically matched and unrelated subjects in the AVNRT cohort and the control group, followed by filtering with VQSR and PLINK1.9 (–geno 0.1 –hwe 0.0001) to obtain high‐confidence variant datasets.2.5n = 82) and the control group (n = 100). We defined deleterious rare  variants as nonsense, missense, splice‐site, indel, and frameshift mutations. For statistical considerations, Fisher's exact test methods were preferred to calculate the gene‐based collapsing. Two groups with MAFs below 0.1% and 1% in the Exome Aggregation Consortium (ExAC) and 1000 Genomes Project databases were calculated separately. A statistical significance was determined by P < 0.05. The significant genes were submitted to the KOBAS3.0 web server (http://kobas.cbi.pku.edu.cn/kobas3) to obtain the functional gene set Reactome Pathway enrichment. Then, the rich factor was calculated, and the top 30 enriched pathways are shown based on the correctedP value.We performed gene‐based collapsing to combine the information on multiple deleterious rare variants into a single value per gene, with ethnically matched and unrelated subjects in the AVNRT cohort (2.6http://string-db.org) was used to predict the relationships among the screened genes and identify the most relevant genes.The Search Tool for the Retrieval of Interacting Genes database (STRING) , nine in RYR3 (n = 9), eight in RYR2 (n = 8), seven in ZFHX3 (n = 7), six in ANK2 (n = 6); five in AKAP9 (n = 5), SYNE2 (n = 5), TRPM4 (n = 5); four in CACNA1D (n = 4), CACNA1I (n = 4), GNB3 (n = 5), MYH6 (n = 4), SCN5A (n = 4); three in HCN4 (n = 3), KCNH2 (n = 3), SCN1A (n = 3), SCN3A (n = 3); two in CACNA1G (n = 2), CACNB2 (n = 2), GJD3 (n = 2), NUP155 (n = 2), SCN4A (n = 2), SCN10A (n = 2), SYNP02L (n = 2); one rare variant in one case in following genes: ADRB2, C9orf3, CASQ2, CAV1, CAV3, ERG, HCN2, HCN3, ITPR1, KCNA4, KCNA5, KCND3, KCNN3, LMNA, PITX2, PKP2, PRKAG2, SCN1B, SCN4B, SCN9A, SLC8A1, SNTA1, SOX5, and TBX3 . Association P values from the GWAS were reported in Q‐Q plots and Manhattan plots  of less than 10−6 are shown in Supplementary Table S4.Single‐nucleotide polymorphisms (SNPs) were removed from the preimputation dataset if they exhibited an MAF < 0.01 or a P < 0.01 according to Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome databases. As shown in Figure 2+ signaling pathway, and (4) ion channel transport, and there were 20 genes in both the KEGG and Reactome database analyses, including ABLIM2, ASPH, ATP2B4, CACNA1G, DPYSL2, EPHA2, FES, MYL12A, NE01, PLXNA4, PLXNB1, PLXNC1, ROBO2, RYR1, RYR2, RYR3, SEMA5A, SEMA6D, SLIT3, and UNC5B  with P < 0.001) . Among the top 30 enriched pathways, there were six related pathways (MAF < 0.01) and two pathways related to AVNRT , SCN1A , CFTR , EPHB4 , PRKAG2 , ROBO1 , ASPH , MMP2 , and ABCC8 . Remarkably, RYR2  was ranked as the first PPI node among the reference genes with rare variants in the present study, and the P value of the burden gene test was nearly 0.05 (P = 0.55) with frequency categories (MAF < 0.001). Considering the functional roles of the genes and previous studies, the most likely candidate genes were SCN1A, PRKAG2, RYR2, CFTR, and NOS1 , we obtained information about 33 rare variants in 18 genes in this database of arrhythmia patients; these genes were SCN1A, PRKAG2, CFTR, NOS1, PIK3CB, GAD2, HIP1R, ASPH, CD163, SLC9B1, ROBO1, EPHB4, KCNV2, PPFIA1, SYT10, COG4, MMP2, and CSF2RB. In particular, rare variants in three genes, PIK3CB, GAD2 and HIP1R, were present even in patients with PSVT  among 791 phenotypes in UK Biobank  in our present study was also found in one Danish AVNRT case, which supports SCN1A and RYR2 gene as candidate pathogenic genes in our study. The detail rare variants information is shown in Table Because the disease information of UK Biobank was not specific enough, we chose the only known AVNRT genetic sequencing study to further validate our candidate pathogenic genes. The study, published in 2018, was carried out in Denmark, and 67 known arrhythmia target genes were detected in AVNRT cases by next‐generation sequencing.4SCN1A, PRKAG2, RYR2, CFTR, NOS1, PIK3CB, GAD2, and HIP1R, responsible for neuronal system/neurotransmitter release or ion channel/cardiac conduction, are likely to be candidate genes and pathways for AVNRT. As this is only a pilot study of the genetic investigation of AVNRT, further genetic functional studies are needed.To our knowledge, this is the first study with the primary aim of investigating the genetic contribution of AVNRT using a WES approach. In the present study and an external data validation, genes such as KCNJ12, RYR3, RYR2, ZFHX3, ANK2, AKAP9, GNB3, SYNE2, CACNA1D, CACNA1I, GNB3, MYH6, SCN5A, SCN1A, SCN3A, HCN4, and KCNH2. Most of these genes, such as KCNJ12, RYR3, RYR2, CACNA1D, CACNA1I, SCN5A, SCN1A, SCN3A, HCN4, and KCNH2, encode ion channels, indicating that AVNRT was associated with ion channels. Interestingly, the causal gene of Wolff‐Parkinson‐White syndrome, PRKAG2, was also identified in our AVNRT cases.Recently, an increasing number of clinical reports have suggested that there may be a hereditary contribution to AVNRT.The autonomic nervous system is known to take part in the triggering and termination of AVNRT.+ current (Ik‐ACh), and inhibit the funny current (If) and the L‐type Ca2+ current.If and the L‐type Ca2+ current and induce changes in intracellular Ca2+ handling.SCN1A) and potassium channels (KCNV2) were selected as candidate genes; in particular, SCN1A is regarded as one of the most likely candidate pathogenic genes. Mutations in ion channel genes might affect the conduction of AV nodes, and differences in conduction velocity will lead to dual AV node physiology and AVNRT.The autonomic nervous system also shows a close relationship with cardiac ionic conductance. Vagal nerve endings release acetylcholine, activate the ACh‐activated Kcurrent Ik‐ACh, ancurrent Ik‐ACh, ancurrent Ik‐ACh, ancurrent Ik‐ACh, ancurrent Ik‐ACh, anPRKAG2 gene, encoding the gamma2 regulatory subunit of adenosine monophosphate‐activated protein kinase, which was identified as the pathogenic gene of Wolff‐Parkinson‐White syndrome.PRKAG2 mutations induce the slowing of sodium channel inactivation and increase the likelihood of channel activation at more negative potentials.SCN1A, which is primarily a neuronal gene. Nav1.1, a product of SCN1A, is present in various regions of the heart.SCN1A mutations are found in up to 80% of patients with Dravet syndrome, a type of epilepsy observed in infancy, and sudden unexpected death results in 38% of all deaths in patients with a childhood onset.SCN1A‐R1407X knock‐in mice,SCN1A variants might cause sudden death or lethal arrhythmia through neurocardiac or solely cardiac mechanisms. The third gene was RYR2, encoding cardiac ryanodine receptors (RYR2s), which are large intracellular Ca2+ channels that regulate the release of Ca2+ from the sarcoplasmic reticulum in cardiomyocytes.RYR2 can increase the probability of channel open during diastole, resulting in excess diastolic SR Ca2+ release, and the increased SR Ca2+ leak during diastole can increase the frequency of spontaneous Ca2+ sparks, resulting in an untimely depolarizing inward current that triggers delayed after depolarization and ventricular arrhythmia or atrial fibrillation.There were three genes with rare variants reported to be associated with arrhythmia among the candidate genes in the present study. The first was the CFTR. It encodes a cAMP‐activated chloride channel  and is presented mainly in epithelial cells of the respiratory and digestive tracts; mutations in this gene cause cystic fibrosis.+ channels, ATP channels, and inward rectifier K+ channels.NOS1. It encodes neuronal nitric oxide synthase (nNOS) and is a major isoform within the brain.Ikr), and shortens action potential,CFTR and NOS1 have not been proven to directly cause arrhythmia, all the potential evidence listed above shows that these gene mutations can change some characteristics of atrioventricular node conduction by affecting the autonomic nervous system/neurotransmitter release or ion channels, which can lead to changes in cardiac depolarization, action potentials, cardiac conduction velocity, the refractory period, etc., and such changes will lead to dual AV node physiology and AVNRT.Among the other two candidate genes, the first was PIK3CB, GAD2, and HIP1R, were present even in patients with PSVT in the UK Biobank resource. However, there was no data indicating the three genes play any role in cardiac arrhythmia. PIK3CB encodes an isoform of the catalytic subunit of phosphoinositide 3‐kinase beta (PI3Kβ), recent data showed PI3K signaling activation affected currents of multiple ion channels, including calcium and sodium channels, and suppression of PI3K activation displayed a prolonged QT interval.GAD2 is a glutamate decarboxylase 2 coding gene, diseases associated with GAD2 include autoimmune polyendocrine syndrome and stiff‐person syndrome, among its related pathways are neurotransmitter release cycle and database. Diseases associated with HIP1R (Huntingtin interacting protein 1 related) include expressive language disorder and cataract. At present, there is a lack of data about mutations of the three candidate genes in arrhythmia or AVNRT, which needs to be confirmed by other large samples genetic research or functional verification.As for the three candidate genes, 5SCN1A, PRKAG2, RYR2, CFTR, NOS1, PIK3CB, GAD2, and HIP1R, in AVNRT in the present study, the genes were not verified experimentally, and further research is needed to explore the potential mechanisms of these genes. Since AVNRT is caused by complex molecular mechanisms, a single pathway is not sufficient to explain the pathogenesis of this disease. Therefore, further experimental research is needed to confirm the current findings. Finally, the controls included in the present study did not undergo invasive electrophysiological examination, and it is possible that the controls were not completely devoid of AVNRT.The major limitation of the current study is the small sample size as AVNRT with low prevalence in population. Although we identified a few candidate genes, such as 6SCN1A, PRKAG2, RYR2, CFTR, NOS1, PIK3CB, GAD2, and HIP1R, in the pathways of neuronal system/neurotransmitter release cycles or ion channel/cardiac conduction, which require further replication in larger cohorts and functional confirmation. Because the anatomic substrate in AVNRT remains unclear, our findings may provide insight into the molecular basis of AVNRT and provide a new view of AVNRT as shown in Figure Our study identified a number of potentially disease‐related genes, such as This work was supported by National Natural Science Foundation of China .The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. The manuscript has been approved by the responsible authorities of the institutions where the work was conducted, and all authors have read the manuscript and approved its submission to your journal.Supporting InformationClick here for additional data file.Supporting Information S1Click here for additional data file.Supporting Information S2Click here for additional data file.Supporting Information S3Click here for additional data file.Supporting Information S4Click here for additional data file.Supporting Information S5Click here for additional data file.Supporting Information S6Click here for additional data file.Supporting Information S7Click here for additional data file.Supporting Information S8Click here for additional data file.Supporting Information S9Click here for additional data file.Supporting Information S10Click here for additional data file.Supporting Information S11Click here for additional data file.Supporting Information S12Click here for additional data file.Supporting Information S13Click here for additional data file.Supporting Information S14Click here for additional data file.Supporting Information S15Click here for additional data file.Supporting Information S16Click here for additional data file.


Aergic synapse density and IPSCs in CA1 pyramidal cells from Tg21 mice is observed. Detection of EPO receptor (EPOR) mRNA was observed to be restricted to glutamatergic pyramidal cells and increased in Tg21 mice at postnatal day (P)7, along with reduced apoptosis. Our findings show that EPO can stimulate postnatal GABAergic maturation in the hippocampus, by increasing neuronal survival, modulating critical plasticity periods, and increasing synaptic transmission. Our data supports EPO’s clinical use to balance GABAergic dysfunction.Several neurodevelopmental disabilities are strongly associated with alterations in GABAergic transmission, and therapies to stimulate its normal development are lacking. Erythropoietin (EPO) is clinically used in neonatology to mitigate acute brain injury, and to stimulate neuronal maturation. Yet it remains unclear whether EPO can stimulate maturation of the GABAergic system. Here, with the use of a transgenic mouse line that constitutively overexpresses neuronal EPO (Tg21), we show that EPO stimulates postnatal GABAergic maturation in the hippocampus. We show an increase in hippocampal GABA-immunoreactive neurons, and postnatal elevation of interneurons expressing parvalbumin (PV), somatostatin (SST), and neuropeptide Y (NPY). Analysis of perineuronal net (PNN) formation and innervation of glutamatergic terminals onto PV+ cells, shows to be enhanced early in postnatal development. Additionally, an increase in GABA Using a mouse model that overexpresses recombinant human erythropoietin (EPO) in the CNS, we observed stimulation of the postnatal maturation of GABAergic transmission in the hippocampus, notably accelerated maturation of parvalbumin (PV)+ interneurons, enhanced glutamatergic inputs onto these interneurons, and enhanced IPSCs onto pyramidal cells. We show that EPO receptors (EPORs) are expressed on pyramidal cells, therefore the impact of EPO on GABAergic maturation is likely to be indirect. Our data show that EPO can modulate hippocampal network maturation and support ongoing trials of the use of EPO in clinical neonatology to stimulate neuronal maturation after perinatal brain injury (PBI).A receptors (GABAARs), and perturbed parvalbumin (PV) and somatostatin (SST) expression in cortical interneurons, have been reported in the neonatal brain after injury  might lead to psychiatric disorders associated with alterations in GABAergic transmission . A signir injury . In addir injury . Perturbr injury . TherefoErythropoietin (EPO), the erythropoietic hormone . is a leARs on the plasma membrane increasing synaptic transmission in neurons (EPO and its receptor [EPO receptor (EPOR)] are expressed in human and mouse brain . Specifi neurons . TherefoDuring the second postnatal week, GABAergic transmission in the CA hippocampus switches from excitatory, because of elevated intracellular chloride concentration, to inhibitory at around postnatal days (P)13–P15 , an age Tg/+, EPORfx/fx mice. Our data show that EPO increases postnatal neuronal survival, enhances GABAARs and conductance onto pyramidal cells; increases glutamatergic inputs onto PV+ cells, and accelerates PNN formation. Thus, EPO is a potential drug to stimulate hippocampal GABAergic maturation without causing any network imbalance.In this work, we quantified the numbers of interneurons immunoreactive for GABA, PV, SST, and neuropeptide Y (NPY), analyzed the formation of PNNs, evaluated the glutamatergic innervation of PV+ neurons, we quantified the density of GABAergic synapses in the CA1 area and recorded spontaneous IPSC (sIPSC) and miniature IPSC (mIPSC) in pyramidal cells using whole-cell electrophysiology. Finally, we determined, the cellular mRNA expression of EPORs in the CA1 area and evaluated EPORs and PV+ cell numbers in GAD65-cread libitum. At least three animals per genotype and age from three different litters were used for each experiment.Animal experiments were performed following the ARRIVE guidelines and were approved by the Cantonal Veterinary Office of Zurich, Switzerland. Mice were bred on a C57BL/6 background at the Laboratory Animal Service Centre of the University of Zurich and kept in standard housing conditions with food and water provided The Tg21 transgenic mouse line overexpressing EPO in the CNS was generated by pronuclear microinjection of the full-length human EPO cDNA driven by the platelet-derived growth factor (PDGF) B-chain promoter into fertilized oocytes derived from B6C3 hybrid mice . The resfx/+ offspring were bred again with homozygous EPOR floxed mice. Cre-positive, EPORfx/fx offspring was then used for immunostaining and fISH analysis and compared with Cre-negative, EPORfx/fx mice.Heterozygous GAD65-Cre  male mice were bred with homozygous EPOR floxed (fx) female mice . Cre-positive, EPORfx/fx and GAD65-cre-negative, EPORfx/fx were collected at P11, and anesthetized by intraperitoneal pentobarbital injection . They were then perfused transcardially with PBS (pH 7.4) to rinse blood, followed by fixative containing 4% paraformaldehyde in 0.15 m Na-phosphate buffer, pH 7.4. Brains were immediately dissected, cut sagittal through the midline, and postfixed in the same fixative for 24 h for P7, 18 h for P11, 12 h for P14, and 3 h for P21 and P60 tissue. After postfixation, brains were transferred to 30% sucrose (in PBS) at 4°C for 24–72 h until tissue sank, for cryoprotection. Brains were cut into sagittal serial sections at 50 μm for P7, and 40-μm thickness for other ages using a sliding blade freezing microtome . Six serial sections were collected for brains at P7, eight serial sections for P11, 10 serial sections for P14, and 12 serial sections for P21 and P60. Sections were stored at −20°C in antifreeze solution until use.WT and Tg21 mice of both sexes were collected at P7, P11, P14, P21, and P60; GAD65-cre-positive, EPORm Na-phosphate buffer, pH 7.4) 3 h for P11 and P14 and 90 min for P21 and P60. Brains were then rinsed twice with cold PBS, transferred to 30% sucrose in PBS for cryopreservation and stored for 3 d at 4°C. Brains were frozen with dry ice and kept at −80°C until further use. Sagittal sections were cut as described above.WT and Tg21 mice of both sexes collected at P11, P14, P21, and P60, were deeply anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg), and perfused transcardially at a constant flow rate with 8–12 ml/min of ice-cold oxygenated artificial CSF (ACSF), pH 7.4. Following immediate decapitation, the brain was dissected, cut sagittal through the midline and postfixed in ice-cold 4% paraformaldehyde  for 5 min under agitation, before the reaction was started by adding 2 ml of DAB solution containing 0.01% hydrogen peroxidase. After 5–7 min, depending on the intensity of the staining, the reaction was stopped by transferring the sections into ice-cold PBS followed immediately by another washing step of three times 10 min in PBS. The sections were mounted on gelatin-coated glass slides and left to dry overnight. On the following day, they were dehydrated in ethanol of increasing concentrations  for 5 min each followed by clearing in xylene four times for 5 min. Finally, the sections were cover-slipped with Eukitt mounting medium (Merck).PV-immunoreactive, SST-immunoreactive, NPY-immunoreactive, and CB-immunoreactive interneurons in the hippocampus were quantified in sections processed for immunoperoxidase staining. Free-floating sections were washed three times for 10 min each with Tris-Triton buffer, pH 7.4 followed by incubation with rabbit primary antibody against PV  in a solWisteria floribunda agglutinin (WFA) coupled to Cy3 for staining of PNNs] in a solution containing 0.5 μl DAPI and 2% NGS in Tris-Triton buffer, pH 7.4 at RT for 30 min in the dark. After another washing step of three times 10 min with Tris-Triton buffer, pH 7.4, sections were mounted on gelatin-coated glass slides and cover-slipped with Dako fluorescence mounting medium (Dako).Double or triple immunofluorescence staining was used to analyze multiple markers within the same section. Free-floating sections were washed three times for 10 min each in Tris-Triton buffer, pH 7.4 before being incubated overnight at 4°C under continuous agitation with the primary antibodies raised in different species  in a solfx/fx, and GAD65-cre-negative, EPORfx/fx mice at postnatal age P11, were deeply anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg) followed by decapitation and dissection of brain tissue on ice. Hemispheres where then frozen on dry ice and stored at −80°C until use. Serial brain sections of 10 μm were cut using a cryostat (Leica Biosystems), mounted on Superfrost slides and stored at −80°C.WT, Tg21 mice of both sexes at postnatal ages P7, P11, P21, and P60, and GAD65-cre-positive, EPORfISH of murine EPOR (RNAscope Probe-Mm-Epor) was performed, using the RNAscope Multiplex Fluorescent reagent kit v2 from Advanced Cell Diagnostics and the fluorophore Opal 520 from PerkinElmer. Positive and negative control probes (RNAscope 3-plex positive and negative control probes) were always run in parallel.m Na-phosphate buffer (pH 7.4), treated 10 min with hydrogen peroxide at RT, target retrieval for 10 min at 85°C  and protease treatment with Protease Plus for 30 min at 40°C. Probes were hybridized for 2 h at 40°C. Slides were washed in Tris-Triton buffer, pH 7.4 followed by amplification steps (RNAscope Amp1) and signal development (HRP-C1 + fluorophore 1). Finally, slides were incubated for 3 min in DAPI and coverslipped.Fresh frozen tissue was postfixed for 30 min at 4°C in 4% paraformaldehyde prepared in 0.15 Sections processed for immunoperoxidase staining were visualized and photographed with an Axioscope 2 microscope (Carl Zeiss AG) equipped with a color digital camera (AxioCam MRc5) and its corresponding software, AxioVision 4.5 (Carl Zeiss AG). Images of the whole hippocampus were taken with bright-field illumination using a 5× objective (NA 0.15).-3 and DAPI samples were imaged with a Zeiss Axio Imager 2 fluorescent microscope (Carl Zeiss AG). Image stacks  from CA3 and CA1 areas were taken with a 10× objective, with a scan zoom of 1× and image size of 1024 × 1024 pixels.Images of cleaved caspasez-stacks  with a scan zoom of 1× for CA3 area and 0.5× for the CA1 area and an image size of 1024 × 1024 pixels.Immunofluorescence GABA and NeuN-stained tissue sections were imaged using a Zeiss LSM 700 confocal laser scanning microscope (Carl Zeiss AG) with a 40× oil immersion objective with a numerical aperture (NA) of 1.4. Images were taken as ARγ2 subunits were stained, and sections were imaged using a Zeiss LSM 800 confocal laser scanning microscope (Carl Zeiss AG). For glutamatergic inputs, vesicular glutamate transporter (vglut)1-2 and PV cells stained sections were imaged. A 63× oil immersed objective with a NA of 1.4 was used for synapse analysis. Images were taken as z-stacks  with a scan zoom of 1.5× and an image size of 1024 × 1024 pixels. Imaging parameters were kept constant over all conditions. After acquisition, images were processed using 2D super resolution Airy Scan processing run in automated mode. image acquisition was conducted in the stratum pyramidale and stratum radiatum of the CA1 and CA3 region.For GABAergic synaptic cluster analysis, vesicular GABA transporter (VGAT), gephyrin and GABAz-intervals of 0.4 μm. Image acquisition was performed in the CA1 and the CA3 area of the hippocampus and image settings were kept constant between genotypes within every age.For analysis of PNNs around PV+ interneurons, double-stained sections were imaged using a Zeiss LSM 800 confocal laser scanning microscope (Carl Zeiss AG) with a 10× objective (NA 0.45) and taking image stacks composed of 22 slices at a Samples were imaged with a Zeiss LSM 700 confocal laser scanning microscope (Carl Zeiss AG). Image stacks  from CA1 and CA3 stratum pyramidale and radiatum were acquired with at 40× objective, N.A. 1.4.Unbiased counting of PV+, SST+, and NPY+ interneurons in the hippocampus was performed using the 10× objective (NA 0.45) of an Axioplan 2 bright-field microscope (Carl Zeiss AG) equipped with a digital camera . First, the hippocampal CA1 and CA3 areas containing the different subregions: stratum oriens, stratum pyramidale, stratum radiatum and stratum lacunosum moleculare, were delineated with Mercator Pro software (Explora Nova) according to the mouse brain atlas . Subsequtot) of the CA1 and CA3 were calculated from the ssf, the areas delineated in every section  and the section thickness (h) as follows:The total volumes (Vtot) in the CA1 and CA3 were calculated using the ssf and the positive cell numbers per section (Qi):Subsequently, the total number of immuno-positive cells (QN) were calculated using the formula: Total number of NeuN+, and GABA+ cells were quantified with the optical Fractionator using the Stereo Investigator software  equipped for fluorescence imaging, with a 63× lens (Zeiss 1.4 Oil). Neu+ and GABA+ cells were counted independently in a frame of 40 × 40 μm with a step size of 120 μm. Total cell numbers . Images were digitzed using a precision illuminator  and CoolSNAP cf. photo-camera (Photometrics) with a Micro-Nikkor (55 + 12 mm) objective (Nikon Corp.). Next, a gray value calibration (Kodak step tablet no. 310ST607) was performed, and the intensity was measured in the different regions of interest (ROIs). To correct for variations in background staining, the intensity value was normalized to the intensity of the corpus callosum. A total of five images per animal and three animals per genotype, were analyzed.z-stacks. Cell densities of caspase-3+ cells were directly quantified from each field of view. All imaging parameters were kept constant between groups. Images were processed with Fiji ImageJ (NIH).Four brain sections were imaged per mouse and maximum intensity projections were created from z-stacks using a custom-made macro in ImageJ (NIH) software. Processing of both channels included thresholding method Yen for PNN and Otsu for PV, background subtraction, Gaussian blurring, and size restrictions. Six animals per age and genotype were used for the analysis. Data points represent average values of the total quantified cells per animal.Raw confocal images were preprocessed to 8-bit grayscale tiff files, before being z-projected into a sum slices image using ImageJ (NIH) software, allowing the image to be analyzed in two dimensions without losing any information. In a next step, PV+ cells and PNN number, size and area were analyzed using the Perineuronal net Intensity Program for the Standardization and Quantification of ECM Analysis (PIPSQUEAK) macro  in FIJI z-stack, using a custom-made macro in ImageJ software (NIH). Processing of all channels included background subtraction with Rolling Ball Radius, Gaussian blurring, thresholding for selecting ROIs of high staining intensity , as well as shape (circularity: 0.4–1) and size  restrictions for cluster detection, using the same parameters in all images per genotype and age. GABAergic synapses were detected based on colocalization of a γ2-GABAAR, gephyrin, and VGAT-immunofluorescence. vGlut1-2 inputs onto PV+ cells were quantified by colocalization of both channels. To determine apposition of presynaptic markers (vGAT and vGluT), the size of the cluster was increased by one pixel all around  software. Processing of EPOR particles (green channel) was separately analyzed, with background subtraction using rolling ball radius, Gaussian blurring, and thresholding for selecting ROIs of high staining intensity, as well as shape (0.5–1 circularity) and size (0.1–1 μm in diameter) restrictions. The same parameters were used in all images per genotype and age. Six animals were used for the analysis.Analysis was done in n(cells) = 8, n = 4 and for Tg21 n(cells) = 9, n = 5; at P19–P22, WT n(cells) = 8, n = 6 and for Tg21 n(cells) = 8, n = 6. The brain was quickly removed and transferred to ice-cold dissection solution containing 65 mm NaCl, 2.5 mm KCl, 1.25 mm NaH2PO4, 25 mm NaHCO3, 7 mm MgCl2, 0.5 mm CaCl2, 25 mm glucose and 105 mm sucrose, saturated with 95% O2 and 5% CO2. 350 μm-thick sagittal slices containing the hippocampus were cut from the tissue block with a vibratome (Leica) and kept in oxygenated ACSF (315 mOsm) containing 125 mm NaCl, 2.5 mm KCl, 1.25 mm NaH2PO4, 25 mm NaHCO3, 1 mm MgCl2, 2 mm CaCl2, and 25 mm glucose at 32°C for 25 min and then at RT until use.WT and Tg21 in the postnatal age ranges of P13–P15 and P19–P22 were briefly anaesthetized with isoflurane and decapitated. The number of recordings for each group are as follows; at P13–P15, WT m NBQX, 50 μm AP-5 and 0.5 μm Strychnine to block glutamatergic and glycinergic transmission. For mIPSCs, 1 μm tetrodotoxin (TTX) was further added to block events caused by action potentials firings. Recordings with an unstable baseline or a holding current greater than −120 pA were rejected. Recording pipettes were made from borosilicate glass , pulled with a vertical puller (Narishige PC-10), had resistances of 7–9 MΩ when immersed in ACSF and filled with internal solution containing the following: 130 mm K-gluconate, 5 mm NaCl, 10 mm HEPES, 1 mm EGTA, 5 mm Mg-ATP, and 0.5 mm Na-GTP; pH 7.4, 290–300 mOsm. sIPSCs and mIPSCs were recorded with an internal solution containing the following: 120 mm CsCl, 4 mm MgCl2, 10 mm HEPES, 10 mm EGTA, 2 mm Mg-ATP, and 0.5 mm Na-GTP; pH 7.4, 290–310 mOsm. Recordings were performed using Multiclamp 700B amplifier (Molecular Devices), data were digitized with Digidata 1440A (Molecular Devices) and acquired with the acquisition software Clampex 10.0 (Molecular Devices). All experiments were performed at RT (21–23°C).For recording, individual slices were transferred to a recording chamber perfused with oxygenated ACSF (as aforementioned) at a flow rate of 1–2 ml/min at RT (21–23°C). Whole-cell patch clamp recordings were made from hippocampal CA1 pyramidal neurons visualized using differential interference contrast (DIC) with an upright microscope  at a low, 10× (water immersion objective) magnification; high, 63× (water immersion objective) magnification was used for approaching the cell and achieving a high-resistance (gigaohm) seal. sIPSCs were recorded from CA1 pyramidal cells, clamped at a holding potential of –70 mV, in the presence of 20 μsIPSCs and mIPSCs currents were filtered off-line using a Butterworth low-pass filter (2 kHz), digitized at 10 kHz and analyzed as 2-min epochs following the addition of the pharmacological blockers for 2 min, using the Mini-Analysis Program 6.0.7 . Recordings with leak increasing >120 pA and access resistance changing >30% between the beginning and the end of the recording were discarded. At least 200 events were analyzed for any condition in all experiments. Synaptic events were identified by setting the event detection threshold at least 2-fold higher than the baseline noise level and by ensuring that events had (1) rise times faster than the decay time, (2) rise times >0.5 ms, and (3) decay times >1.5 ms; only events adhering to these parameters were included in further analysis. Event amplitudes, interevent intervals, rise and decay times were averaged within each experiment. The frequencies were calculated from the interevent intervals and the resulting means were averaged between experiments.t test. Data in different brain areas that were influenced by one factor were analyzed with multiple t test. To compare data influenced by two factors, a two-way ANOVA with Bonferroni multiple comparisons post hoc test was used. Probability distributions were tested using the Kolmogorov–Smirnov (KS) test. For all statistical tests, p < 0.05 was considered statistically significant. Data are presented as mean ± SD. In order to confirm appropriate sample sizes for experiments, a Power Analysis was conducted using G*Power software . Parametric data with two conditions were analyzed using an unpaired, two-tailed Student’s F = 16.3, p = 0.0002; F = 77.90, p < 0.0001;F = 16.53, p = 0.0002;To determine whether constitutive overexpression of human EPO in the CNS influences GABAergic neurotransmission in the hippocampus, the total number of neurons (immunoreactive for NeuN) and the total number of GABA-immunoreactive neurons were stereologically quantified together in the CA3 and CA1 regions of Tg21 mice at P7, P14, P21, and P60 and compared with the WT control mice A,B. Reprt test, p = 0.024 and p = 0.044, respectively;t test, p = 0.005 and p = 0.018, respectively;To determine whether this early increase of GABA immunoreactivity at P14 occurs in specific subpopulations of interneurons, we quantified the total number of PV+, SST+, and NPY+, interneurons in CA1 and CA3 at this age C,D. The t test, p = 0.007;t test, p = 0.046;p = 0.0003;F = 32.43, p < 0.0001;F = 21.29, p < 0.0001;Given the importance of PV+ cells for the regulation of pyramidal cell activity and synchronization during postnatal development, we further explored the effect of EPO on PV+ cell numbers, focusing on the CA1 and CA3 areas at P7, P11, P14, P21, and P26 . These tTaken together, our results point to an accelerated maturation of GABAergic neurons in the hippocampus, along with a neurotrophic effect resulting in higher numbers of all neurons persisting until reaching adulthood in Tg21 mice.F = 11.29, p = 0.003;Because EPO is an antiapoptotic cytokine  and manyF = 8.71, p = 0.0048 and CA1: two-way ANOVA, F = 18.36, p < 0.0001;F = 3.8, p = 0.049 and CA1: two-way ANOVA, F = 3.78, p = 0.049;r), p < 0.01;F = 8, p = 0.01; CA1, two-way ANOVA, F = 25.26, p < 0.0001;F = 4.893, p = 0.0388;F = 9.814, p = 0.0052; CA1, two-way ANOVA, F = 21.7, p = 0.0002;F = 5.342, p = 0.037;To determine whether EPO increases PV+ cell maturation and synapse stabilization, we evaluated the formation of PNNs around PV+ cells, at P7, P11, P14, P21, and P26 in Tg21 and WT mice. The plant lectin WFA, which selectively binds to ECM glycoproteins in the PNNs, was visualized together with PV immunofluorescence . The folAR γ2-subunit, which is ubiquitous in GABAergic postsynaptic densities, and the scaffolding protein gephyrin, which closely interacts with GABAARs  in the CA1 stratum pyamidale and dendritic synapses in the CA1 stratum radiatum at P14, P21, and P60. Synapses were identified by triple immunofluorescence staining for the VGAT as a marker of presynaptic terminals, the GABA GABAARs .t test, p = 0.0105;C, t test, p = 0.047, D, t test, p = 0.0050), although VGAT staining did not reach statistical significance  detected by confocal laser scanning microscopy was considered to represent individual GABAergic synapses, which were quantified accordingly. At P14, a 2-fold increase in cluster density was observed in the Tg21 mice as compared with WT , suggesting that EPO mainly affects GABAergic synapses from PV interneurons which occur preferentially on the soma and proximal dendrites of pyramidal cells.The increase in synaptic GABAergic markers in CA1 pyramidal cells at P14 raised the possibility that this morphologic change has a functional correlate. We measured sIPSCs and action potential-independent miniature (mIPSCs). Since we obtained very similar findings for both populations of IPSCs, only the results from the mIPSC analysis are presented here .p < 0.01;t test, p < 0.001); as well as larger amplitudes  of EPOR mRNA at postnatal ages P7, P11, P21, and P60. In both genotypes, EPOR expression was found in pyramidal cells of the CA1 area A,B and ifx/fx) in the GAD65-CreTg/+, EPORfx/fx mouse line. In this line, EPOR expression was measured by fISH analysis at P11 (age when EPORs are highly incremented) in CA1 stratum pyramidale. Similar numbers of labeled puncta were detected in GAD65-CreTg/+, EPORfx/fx and control GAD65-Cre+/+, EPORfx/fx  = 0.036, p = 0.8523;To further ensure that EPORs are mainly expressed in principal cells, fISH of EPOR mRNA along with immunolabeling of PV+ cells was performed but no EPORs were found on PV+ cell in the hippocampus. Furthermore, the lack of EPOR expression on GABAergic cells was confirmed with the conditional deletion of EPOR . Therefore, we tested the hypothesis that glutamatergic input might be increased in PV+ interneurons in Tg21 mice. To this end, we used double immunofluorescence for PV and the two VGluT1 and VGluT2 in the CA1 and CA3 area at P14, the postnatal age where most changes in GABAergic transmission were observed . OverallThus, in summary, we conclude that the effect of EPO on GABAergic postnatal maturation results from pyramidal regulation of interneuron network formation and survival.fx/fx mice, additionally suggests pyramidal cells to regulate interneuron survival , increased GABAergic synapse density and function in pyramidal cells, faster maturation of PV+/WFA+ cells, and increased innervation of PV+ interneurons by glutamatergic terminals. All these changes suggest enhanced GABAergic function during a critical period for the proper formation of brain circuits. EPO overexpression in neurons caused a reduction in apoptosis in the CA1 and CA3 hippocampus during the first postnatal week, when EPORs were upregulated in principal cells of the Tg21 mice. Therefore, survival of interneurons during this postnatal window is essential for postnatal development and circuit formation. The lack of EPORs on interneurons, confirmed by the lack of a phenotype observed in the Gad65-cre, EPORsurvival . Our resWe showed an increase in GABAergic neurons  during postnatal hippocampal development caused by a decrease in apoptosis during the first postnatal week. The reduction in apoptosis was mainly observed in the stratum pyramidale of CA3 and CA1 areas and in the CA1 stratum oriens, which compress oriens-lacunosum moleculare interneurons (O-LM) that express SST and NPY. The restricted location of cell somata in the CA1 stratum oriens dictates that the source of excitatory inputs is mainly from pyramidal cells, therefore survival and enhancement of glutamatergic inputs is also expected to occur in SST and NPY cells. At P14 PV-expressing cells are increased mainly in the CA3 area, whereas SST-expressing cells were increased in the CA1 area. Most cells in the CA1 stratum oriens showed reduced apoptosis at P7, therefore the increase in SST number could be linked to survival.AR γ2 subunit, resulting in increased GABAAR trafficking to the postsynaptic membrane and facilitation of GABAergic inhibitory transmission  pathway , inhibitIn summary, our data provide evidence that EPO accelerates the maturation of the GABAergic system in the neonatal (P7–P14) hippocampus, without causing network imbalance. The results support the use of EPO as a therapeutic agent to stimulate normal brain development after PBI.