Invitro System Research Articles

Glucocorticoids inhibit human hematopoietic stem cell differentiation toward a common ILC precursor

Innate lymphoid cells (ILCs) comprise cytotoxic natural killer (NK) cells and helper ILCs (hILCs). Human hILC development is less characterized as compared with that of NK cells, although all ILCs are developmentally related. It has been reported that the immunosuppressive drugs glucocorticoids (GCs) regulate ILC function, but whether they control ILC differentiation from hematopoietic stem cells (HSCs) is unknown. This study sought to analyze the effect of GCs on ILC development from HSCs. This study exploited an invitro system to generate and expand from peripheral blood HSCs a multipotent CD56+ ILC precursor able to differentiate into NK cells, ILC1s, and ILC3s. We also analyzed exvivo, at different time points, the peripheral blood of recipients of allogeneic HSC transplantation who were or were not treated with GCs and compared ILC subset reconstitution. Invitro, GCs favor the generation of NK cells from myeloid precursors, while they strongly impair lymphoid development. In support of these data, recipients of HSC transplantation who had been treated with GCs display a lower number of circulating hILCs, including the ILC precursor (ILCP) previously identified as a systemic substrate for tissue ILC differentiation. GCs impair the development of the CD117+ ILCP from CD34+ HSCs, while they do not affect the further steps of ILCP differentiation toward NK cells and hILC subsets. This reflects an association of GC treatment with a marked reduction of circulating hILCs in the recipients of HSC transplantation.

An in vitro system to silence mitochondrial gene expression

The human mitochondrial genome encodes thirteen core subunits of the oxidative phosphorylation system, and defects in mitochondrial gene expression lead to severe neuromuscular disorders. However, the mechanisms of mitochondrial gene expression remain poorly understood due to a lack of experimental approaches to analyze these processes. Here, we present an invitro system to silence translation in purified mitochondria. Invitro import of chemically synthesized precursor-morpholino hybrids allows us to target translation of individual mitochondrial mRNAs. By applying this approach, we conclude that the bicistronic, overlapping ATP8/ATP6 transcript is translated through a single ribosome/mRNA engagement. We show that recruitment of COX1 assembly factors to translating ribosomes depends on nascent chain formation. By defining mRNA-specific interactomes for COX1 and COX2, we reveal an unexpected function of the cytosolic oncofetal IGF2BP1, an RNA-binding protein, in mitochondrial translation. Our data provide insight into mitochondrial translation and innovative strategies to investigate mitochondrial gene expression.

58 Reduced nutrient availability during invitro culture improves embryo production and morphological quality and alters metabolic status of bovine embryos

Invitro production (IVP) of embryos is designed to reproduce an environment that resembles the female reproductive tract. However, the system does not perform optimally in terms of quality and embryo production. A major setback lies in the loss of dynamics observed in a static invitro system, which might affect the availability of substrates that reach the embryo. A reduction in the amount of nutrients in media has been used as an approach to improve IVP (Ermisch et al. 2020 Sci. Rep. 10, 9263; https://doi.org/10.1038/s41598-020-66019-4). The present study aimed at describing a defined sequential medium (embryonic culture supplementation, ECS) and to investigate the effect of reducing nutrient availability on embryo production, quality, and metabolism. ECS was developed in our laboratory and is a serum-free, salt-based culture medium supplemented with the amount of energy substrates and amino acids found in bovine oviduct (Ov) and uterus (Ut) fluids as previously described (Hugentobler et al. 2007Mol. Reprod. Dev. 74, 445–454; https://doi.org/10.1002/mrd.20607; Hugentobler et al. 2008Mol. Reprod. Dev. 75, 496–503; https://doi.org/10.1002/mrd.20760). Embryos were cultured according to the following ECS supplementation: ECS100 (supplemented with 8mg mL−1 bovine serum albumin and 100% of the energy substrates and amino acids concentrations of the Ov and Ut fluids) and ECS50 (half dilution of ECS100). Bovine oocytes from abattoir ovaries were submitted to IVP using standard protocols. On Day 0 of invitro culture, presumptive zygotes were randomly divided into groups ECS100-Ov or ECS50-Ov. On Day 4, embryos were respectively transferred to ECS100-Ut and ECS50-Ut. Expanded blastocysts were collected on Day 7 to assess embryo production, morphology (total cell number by Hoescht 33342 staining; inner cell mass and trophectoderm cells by CDX2 immunostaining), and metabolic status (mitochondrial activity and reactive oxygen species content by MitotrackerTM RedCMXRos and CellROXTM Green staining, ThermoFisher Scientific; NADH and FAD+ by autofluorescence). Data were analysed by Student’s t-test (a=4%). Although cleavage rates were similar between ECS50 and ECS100 (78.13±3.73 vs. 79.70±4.18; P=0.788), blastocyst rates were positively influenced by the reduction in concentration (28.88±1.74 vs. 16.73±2.41; P=0.004). This difference likely comes from a blockage at the morula stage in group ECS100, because the conversion from morula to blastocyst was 20% lower in this group (57.73±3.81 vs. 38.15±3.45; P=0.008). In terms of morphology, blastocysts produced in ECS50 had a higher number of cells (152.4±9.61 vs. 118.3±7.22; P=0.036), which is explained by the higher number of trophectoderm cells. Finally, metabolic status was affected by nutrient reduction: embryos from ECS50 had higher mitochondrial activity, reactive oxygen species content (P<0.0001), and lower NADH (P=0.01), suggesting higher oxidative phosphorylation to produce energy, as expected at this stage. In conclusion, ECS is a functional medium, and a reduced nutrient concentration (ECS50) improves embryo production, morphological quality, and metabolic status of blastocysts, suggesting that culture conditions must be adapted to the invitro system rather than resembling invivo conditions. This research was funded by FAPESP (2016/00350-0, 2017/18384-0).

45 Embryonic disc development invitro in ovine embryos

Embryonic mortality during the second week of pregnancy has an important economic impact on farming. At this time, the embryo undergoes critical developmental events that cannot be recapitulated invitro, limiting our understanding of these pregnancy losses. After the blastocyst stage, the hypoblast migrates to cover the inner surface of the embryo and the epiblast forms a flat embryonic disc (ED) that initiates gastrulation. The aim of this study was to develop an invitro culture system to support sheep embryo development after the blastocyst stage. Day 6/7 invitro-produced blastocysts were cultured over agarose gels to prevent attachment and allocated to different media: synthetic oviductal fluid with 10% fetal bovine serum (SOF-FBS, n=52), an invitro culture medium (hIVC, n=35) supporting ED formation in human embryos (Deglincerti et al. 2016 Nature 533, 251-254; https://doi.org/10.1038/nature17948), and chemically defined N2B27 medium (n=38) supporting ED formation in bovine embryos (Ramos-Ibeas et al. 2020 Reproduction 160, 579-589, https://doi.org/10.1530/REP-20-0243). At Day 14, survival and embryo area were recorded, the abundance of transcripts encoding interferon Tau (TP1) and metabolic enzymes was analysed by RT-qPCR, and the development of epiblast and hypoblast was assessed by immunostaining for SOX2 and SOX17. Embryo survival and size and the percentage of embryos achieving complete hypoblast migration were significantly reduced in SOF-FBS (Chi-squared test and one-way ANOVA; P<0.05). Only N2B27 medium supported epiblast survival in 11/28 embryos. TP1 expression increased at Day 14 in all culture conditions and metabolism-related genes revealed a shift from anaerobic glycolysis to oxidative phosphorylation after culture in hIVC and N2B27. Next, to promote epiblast development, we allocated blastocysts to N2B27 medium supplemented with activin A (n=45), rho-associated protein kinase (ROCK) inhibitor (ROCKi, n=42), or insulin growth factor 1 (IGF1, n=29). IGF1 reduced significantly the percentage of embryos showing an ED-like structure, whereas activin A supplementation significantly increased epiblast survival (Chi-squared test; P<0.05) and SOX2+ cell number was higher in embryos cultured with ROCK inhibitor. When we combined activin A and ROCKi supplementation (N2B27+A+R, n=151), SOX2+ cell number and the percentage of embryos showing an ED-like structure increased significantly (165.1±53.1 and 31/35 embryos with SOX2+ epiblast cells; ∼89%) compared with N2B27 alone (49.4±12.7 and 5/11; ∼45%) (one-way ANOVA and Chi-squared test; P<0.05). Moreover, 18/31 (∼58%) ED-like structures developed in N2B27+A+R lost the Rauber’s layer (polar trophoblast), and BRACHYURY expression, denoting the onset of gastrulation, was observed in 3/14. In conclusion, we have developed a culture system that supports complete hypoblast migration and ED development invitro, which represents a valuable tool to explore early embryo mortality in livestock species without the need for experimental animals.

Engineered Human Umbilical Cord Derived Erythroid Progenitor Cells (HUDEP2) with Sickle or β-Thalassemia Mutation: An in-Vitro System for Testing Pharmacological Induction of Fetal Hemoglobin

Introduction: Sickle cell disease (SCD) and β-thalassemia are inherited blood disorders caused by mutations in the β-globin gene (HBB). Elucidation of the multiple pathophysiologic mechanisms in SCD and β-thalassemia has resulted in an increasing efforts to identify new treatment modalities to ameliorate the consequences of the disease. However, no consistent in vitro system exists for studies of pharmacological therapies for the diseases. Human umbilical cord-derived erythroid progenitor cells (HUDEP2) are an immortalized CD34+ hematopoietic stem cell-derived erythroid precursor cell line that can differentiate into red blood cells. Here, we engineered sickle HUDEP2 and β-thalassemia HUDEP2 clonal lines through CRISPR/Cas9-mediated editing of the human HBB. We sought to establish if these engineered cell lines exhibit disease phenotypes, and if upon in vitro erythroid differentiation they produce fetal hemoglobin (HbF) in response to hydroxyurea, the only FDA-approved drug for HbF induction. Our goal is to create an in vitro system to test new HbF inducers for treating SCD or β-thalassemia.Materials and Methods: We delivered Hi-Fidelity Streptococcus pyogenes (Sp) Cas9 protein and CRISPR guide RNA as a ribonucleoprotein complex in conjunction with a single-stranded DNA donor (ssODN) template to introduce the sickle or K17X (A<T) or codon 6 [-G] β-thalassemia mutation into the HBB locus of HUDEP2 cells. Edited HUDEP2 cells were single-cell sorted into multiple 96-well plates and expanded. The genotype of the clones was determined using a probe-based droplet digital PCR assay and confirmed through Sanger sequencing. Native polyacrylamide gel electrophoresis and high-performance liquid chromatography (HPLC) were used to confirm the hemoglobin phenotype. Normal parental cell line, sickle clone, and two individual β-thalassemia clones were used to test the pharmacological induction of HbF. We initiated drug treatment in the expansion phase with 30 µM hydroxyurea. Trypan Blue staining and CD71/CD233/CD235 staining determined the effect of the drugs on the viability, growth rate and erythroid development of HUDEP2 lines. After 10 days of drug treatment, differentiated HUDEP2 were analyzed for globin expression through RT-qPCR and HPLC, and HbF positive cells (F-cells) were quantified via flow cytometry. Cells were placed at 2% O2 for four hours, fixed in glutaraldehyde, stained, and viewed under magnification to assess sickling potential.Results and Discussion: We generated multiple clones with biallelic sickle or β-thalassemia mutations. Sickle HUDEP2 clones almost exclusively expressed sickle hemoglobin with low level of HbF and hemoglobin A2 (HbA2), and β-thalassemia HUDEP2 clones produced no normal adult hemoglobin, 8-10% HbF, and 26-28% HbA2. On HPLC analysis, β-Thalassemia HUDEP2 clones had an unknown tall peak (39-45%) between HbF and HbA consistent with an α-globin homotetramer (α4). When subjected to hypoxic conditions for 4 hours, sickle HUDEP2 produced sickle cells. HUDEP2 parent cells did not sickle under hypoxic conditions. Hydroxyurea induced 3.8-fold, 1.8-fold, and 1.6-fold increases in γ-globin gene (HBG) expression; 2.9-fold, 1.4-fold, and 1.4-fold increases in the percentages of F-cells; 1.4-fold, 1.2-fold, and 1.6-fold increase in the percentages of HbF in sickle, K17X(A<T) and codon 6[-G] β-thalassemia HUDEP2 clones, respectively. No change was observed in CD71/CD235 positive HUDEP2 cells in the presence hydroxyurea. This finding demonstrated that hydroxyurea treatment induces HBG expression as well as HbF and F-cells in engineered sickle and β-thalassemia HUDEP2 clones. Future work will include screening other pharmacological compounds as well as studying the mechanism of HbF induction by using HUDEP2 clones.Conclusions: Our engineered sickle and β-thalassemia HUDEP2 cell lines have properties similar to those of patient erythroid cells and respond to the known HbF inducer hydroxyurea. This in vitro model system may facilitate the drug-discovery process by enabling multimodal drug screening on a large scale with consistent and reproducible results.Acknowledgments: This work was supported by the Cancer Prevention and Research Institute of Texas grants RR140081 and RP170721 (to G.B.) and the National Heart, Lung and Blood Institute of NIH (1K08DK110448 to V.S.) DisclosuresNo relevant conflicts of interest to declare.

Sickle Human Umbilical Cord Derived Erythroid Progenitor Cells (S-HUDEP2): An Ideal in-Vitro System for Screening Anti-Sickling Compounds for Sickle Cell Disease

Background: Sickle Cell Disease (SCD) is a devastating inherited disease, characterized by polymerization of sickle hemoglobin under deoxygenated conditions that can lead to acute pain crises, ischemia, and chronic organ damage. Pharmacologic anti-sickling agents that decrease polymerization are currently under investigation, however there is no consistent in vitro system to study these compounds; whole patient blood, subject to clinical variability and limited supply, is most often used. Human Umbilical Cord Derived Erythroid Progenitor 2 cells (HUDEP2) are an immortalized CD34+ hematopoietic stem cell (HSC) derived erythroid precursor cell line that can differentiate into red blood cells. We have engineered S-HUDEP2 cells to express sickle hemoglobin (HbS) via CRISPR/Cas9 gene editing. We hypothesized that this cell line will sickle under hypoxic conditions, produce dense red blood cells (DRBC, red cells with a density>1.11 mg/mL that are dehydrated and prone to sickling. If these intrinsic, essential SCD RBC properties are found, we propose to use this novel cell line to screen drug compounds for anti-sickling capabilities.Methods: S-HUDEP cells were cultured as previously described (Kurita et al, 2013). %HbS and %HbA produced by parent HUDEP and S-HUDEP2 cells were measured by high performance liquid chromatography (HPLC). Hypoxia was induced by placing the cells at 2% O2 for four hours. Parent HUDEP and S-HUDEP2 cells were then fixed with glutaraldehyde and Giemsa stained. % sickling estimated at 40x magnification by a pathologist blinded to cell group counting sickle forms out of 1000 cells. The percentage of dense red blood cells (DRBCs) was quantified by an ADVIA hematology analyzer (Siemens). S-HUDEP2 cells were dosed with 0, 2.5 and 5 µM 5-hydroxymethylfurfural (5-HMF) and 75, 150, and 300 µM GBT440, two known anti-sickling agents, on day ten and day 14 of culture for one hour, subjected to hypoxic conditions and % sickling quantified as described above.Results: S-HUDEP2 cells express 98% HbS. Under hypoxia, 20% of S-HUDEPs sickle at day 10 of differentiation; 30% of S-HUDEP2 cells sickle at Day +14 of differentiation. Parent HUDEP-2 cells, which produce 98% HbA, did not sickle under hypoxic conditions at any stage of differentiation. 70% of S-HUDEP2 cells were determined to be DRBC under hypoxia at Day 10 and 14 time points; parent HUDEP-2 cells did not produce DRBC under hypoxia. Treatment of S-HUDEP2 cells with 5μM of 5-HMF and 150 μM of GBT440 reduced sickling by 40-50% under hypoxic conditions compared with untreated S-HUDEP2 cells (p<0.01), and reduced %DRBC by 30% (p<0.01).Conclusions: S-HUDEP2 cells express HbS, form DRBC, and sickle under hypoxic conditions, just like erythroid precursors and mature red blood cells from individuals with SCD. Exposure to two anti-sickling agents, one of which is currently in Phase III clinical trials, significantly decreased S-HUDEP2 sickling under hypoxic conditions, and reduced %DRBC, a marker of disease severity. Advantages of S-HUDEPs over patient samples include genetic and phenotypic uniformity, no human pathogens, and availability to groups without access to patient samples. We therefore conclude that S-HUDEP2s have utility in clinical research, may be used to screen anti-sickling and anti-dense cell compounds in vitro, and may lead to identification of new therapeutic options for SCD patients. DisclosuresNo relevant conflicts of interest to declare.

Loss of KLF15 accelerates chronic podocyte injury.

Krüppel‑like factor15 (KLF15), also known as kidney‑enriched transcription factor, is known to participate in podocyte differentiation. However, the role of KLF15 in chronic podocyte injury remains incompletely understood, particularly in proteinuric disease models. In the present study, the 5/6 nephrectomy mouse model was used to induce chronic podocyte injury. Human primary podocytes were isolated by flow cytometry and cultured to emulate the injury process in an invitro system. Biopsied kidney tissue samples were obtained from patients with primary membranous nephropathy or diabetic nephropathy in order to analyze the relationship between glomerular KLF15 expression and subsequent outcomes. When 5/6 nephrectomy was predisposed to progressive kidney damage, fibrosis markers increased, while podocyte KLF15 expression decreased. In addition, increased fibrosis marker expression in human primary podocytes following treatment with transforming growth factor‑β was aggravated by the knockdown of KLF15. These trends were reversed after cultured podocytes were treated with cyclosporine. When patients were grouped according to KLF15 expression levels in kidney tissue, the low expression groups were demonstrated to have worse renal outcomes, such as non‑remission of disease and end‑stage renal disease. In conclusion, the present findings revealed that low expression of KLF15 was associated with chronic podocyte injury.

Prion-like Propagation of α-Synuclein Is Regulated by the FcγRIIB-SHP-1/2 Signaling Pathway in Neurons

Recent evidence of prion-like propagation of α-synuclein (α-syn) into neighboring neurons set up a paradigm to elucidate the mechanism of progression of Parkinson's disease (PD) and to develop therapeutic strategies. Here, we show that FcγRIIB expressed in neurons functions as a receptor for α-syn fibrils and mediates cell-to-cell transmission of α-syn. SHP-1 and 2 are activated downstream by α-syn fibrils through FcγRIIB and play an important role in cell-to-cell transmission of α-syn. Also, taking advantage of a co-culture system, we show that cell-to-cell transmission of α-syn induces intracellular Lewy body-like inclusion body formation and that the FcγRIIB/SHP-1/2 signaling pathway is involved init. Therefore, the FcγRIIB-SHP-1/-2 signaling pathway may be a therapeutic target for the progression of PD. The invitro system is an efficient tool for further high-throughput screening that can be used for developing a therapeutic intervention in PD.

Reconstituting Corticostriatal Network on-a-Chip Reveals the Contribution of the Presynaptic Compartment to Huntington’s Disease

Huntington's disease (HD), a devastating neurodegenerative disorder, strongly affects the corticostriatal network, but the contribution of pre- and postsynaptic neurons in the first phases of disease is unclear due to difficulties performing early subcellular investigations invivo. Here, we have developed an on-a-chip approach to reconstitute an HD corticostriatal network invitro, using microfluidic devices compatible with subcellular resolution. We observed major defects in the different compartments of the corticostriatal circuit, from presynaptic dynamics to synaptic structure and transmission and to postsynaptic traffic and signaling, that correlate with altered global synchrony of the network. Importantly, the genetic status of the presynaptic compartment was necessary and sufficient to alter or restore the circuit. This highlights an important weight for the presynaptic compartment in HD that has to be considered for future therapies. This disease-on-a-chip microfluidic platform is thus a physiologically relevant invitro system for investigating pathogenic mechanisms and for identifying drugs.

Multi‑target inhibition by four tandem shRNAs embedded in homo‑ or hetero‑miRNA backbones.

The functional influence of microRNA (miRNA)backbone selection remains unclear with respect to multiplexing miRNA‑based short hairpin RNAs (shRNAmiRs), due to a lack of comparative studies. To this end, a pair of shRNAmiR tetramers were designed in the present study that targeted four genes with a shared miR30a backbone (homo‑BB) or four miRNA backbones (hetero‑BB). A PBLT+ 293A cell line overexpressing four targets was established, which permitted simultaneous dissection of individual gene knockdown. Multi‑target inhibition was confirmed by a decrease in positive cell populations of the relative gene and mean fluorescence intensities, with almost comparable activities of homo‑ and hetero‑BB tetramers. Of note, this multi‑inhibition was sustained over a 1‑month period, with no notable difference, particularly in the late‑phased inhibitory effects between homo‑ and hetero‑BB tetra‑shRNA miRs. These preliminary data may indicate little influence of scaffold substitution in the functionalities of multiplexed shRNAmiRs and little recombination‑depleted risk of repetitively adopting the same miRNA backbone in this artificial invitro system. More comparative studies are further required to explore extended repertoires of scaffold‑paralleled multi‑shRNAmiRs in more physiologically relevant models.

Evaluation of an invitro system to simulate equine foregut digestion and the influence of acidity on protein and fructan degradation in the horse's stomach.

The aim of this study was to improve an invitro system in order to gather optimized information on the digestion of different forages in the horse's upper gastrointestinal tract. Therefore, foregut digestion of several forages was simulated invitro (Part 1). The effect of different pH values on invitro fructan degradation of two selected grasses (Part 2) was tested subsequently. Part 1: We hypothesized that our system produces representative results simulating digestive processes in the upper alimentary tract, but neglects microbial fermentation. In vitro digestion of six forages (grass mixture for horses, grass mixture for cows (GMC), tall fescue, English perennial ryegrass (ER), white clover, lucerne) was performed in two phases with pepsin and pancreatin. The results are consistent with current data from invivo studies, including a degradation of crude protein and monosaccharides as well as a relative increase in fibres. Interestingly, a loss of fructan was measured in two feedstuffs (ER/GMC: 4.1/4.4% DM fructan before and 0.59/0.00% DM after simulated foregut digestion). Part 2: As fructans are thought not to be fragmented by digestive enzymes, another hypothesis was developed: acidic hydrolysis leads to a degradation of fructans. To evaluate the influence of gastric pH on the digestion of fructan and protein, different pH values (2, 3 and 4) were adjusted in a second series of invitro foregut digestion trials with ER and GMC. As expected, the highest degradation of protein was seen at the lowest pH (protein in ER/GMC at pH 2: 6.11/8.28% DM and at pH 4: 7.73/10.64% DM), whereas fructan degradation was highest at pH 4 (fructan in ER/GMC at pH 2: 1.63/1.95% DM and at pH 4: 1.31/0.91% DM). We presume that not only acidic hydrolysis but also plant enzymes cause the loss of fructans in an acidic environment.

Interactions between integrase inhibitors and human arginase 1.

The neuro-pathogenic mechanism(s) underlying HIV-associated neurocognitive disorders are mostly unknown. HIV-infected macrophages and microglial cells play a crucial role and the metabolic fate of l-arginine may be highly relevant to microglia activation. In this context, arginase (ARG), which uses l-arginine as substrate, can be on the same time a target and source of oxidative stress and inflammation. In this study, we investigated whether integrase strand transfer inhibitors share with the other antiretroviral drugs the ability to inhibit ARG activity. We used the previously validated cell model, namely the human microglia cell line, as well as the computational chemistry approach. Furthermore, here we characterized the activity of purified human ARG in a cell-free invitro system, and investigated the effects of integrase strand transfer inhibitors in this newly validated model. Overall evidence shows that Dolutegravir, Raltegravir and Elvitegravir inhibit ARG activity.

Dendritic Cells Interpret Haptotactic Chemokine Gradients in a Manner Governed by Signal-to-Noise Ratio and Dependent on GRK6

Navigation of cells along gradients of guidance cues is a determining step in many developmental and immunological processes. Gradients can either be soluble or immobilized to tissues as demonstrated for the haptotactic migration of dendritic cells (DCs) toward higher concentrations of immobilized chemokine CCL21. To elucidate how gradient characteristics govern cellular response patterns, we here introduce an invitro system allowing to track migratory responses of DCs to precisely controlled immobilized gradients of CCL21. We find that haptotactic sensing depends on the absolute CCL21 concentration and local steepness of the gradient, consistent with a scenario where DC directionality is governed by the signal-to-noise ratio of CCL21 binding to the receptor CCR7. We find that the conditions for optimal DC guidance are perfectly provided by the CCL21 gradients we measure invivo. Furthermore, we find that CCR7 signal termination by the G-protein-coupled receptor kinase 6 (GRK6) is crucial for haptotactic but dispensable for chemotactic CCL21 gradient sensing invitro and confirm those observations invivo. These findings suggest that stable, tissue-bound CCL21 gradients as sustainable "roads" ensure optimal guidance invivo.

Xenografting of isolated equine (Equus caballus) testis cells results in de novo morphogenesis of seminiferous tubules but not spermatogenesis.

The study of spermatogenesis in the horse is challenging because of the absence of an invitro system that is capable of reproducing efficient spermatogenesis and because of the difficulties and costs associated with performing well-controlled studies invivo. In an attempt to develop novel methods for the study of equine spermatogenesis, we tested whether cells from enzymatically digested pre-pubertal equine testicular tissue were capable of de novo tissue formation and spermatogenesis following xenografting under the back skin of immunocompromised mice. Testes were obtained from normal pre-pubertal colts and dissociated into cell suspensions using trypsin/collagenase digestion. Resulting cell pellets, consisting of both somatic and germ cells, were injected into fascial pockets under the back skin of immunocompromised, castrated mice and maintained for between 1 and 14months. Mice were killed and grafts were recovered and analyzed. As has been reported for testis cell suspensions from pigs, mice, cattle, and sheep, de novo formation of equine testicular tissue was observed, as evidenced by the presence of seminiferous tubules and an interstitial compartment. There was an increased likelihood of de novo testicular formation as grafting period increased. Using indirect immunofluorescence, we confirmed the presence of spermatogonia in de novo formed seminiferous tubules. However, we found no evidence of meiotic or haploid cells. These results indicate that dissociated pre-pubertal equine testis cells are capable of reorganizing into the highly specialized endocrine and spermatogenic compartments of the testis following ectopic xenografting. However, in spite of the presence of spermatogonia within the seminiferous tubules, spermatogenesis does not occur. Although this technique does allow access to the cells within the seminiferous tubule and interstitial compartments of the equine testis prior to reaggregation, the absence of spermatogenesis will limit its use as a method for the study of testicular function in the horse.

The Histone Modification Domain of Paf1 Complex Subunit Rtf1 Directly Stimulates H2B Ubiquitylation through an Interaction with Rad6

The five-subunit yeast Paf1 complex (Paf1C) regulates all stages of transcription and is critical for the monoubiquitylation of histone H2B (H2Bub), a modification that broadly influences chromatin structure and eukaryotic transcription. Here, we show that the histone modification domain (HMD) of Paf1C subunit Rtf1 directly interacts with the ubiquitin conjugase Rad6 and stimulates H2Bub independently of transcription. We present the crystal structure of the Rtf1 HMD and use site-specific, invivo crosslinking to identify a conserved Rad6 interaction surface. Utilizing ChIP-exo analysis, we define the localization patterns of the H2Bub machinery at high resolution and demonstrate the importance of Paf1C in targeting the Rtf1 HMD, and thereby H2Bub, to its appropriate genomic locations. Finally, we observe HMD-dependent stimulation of H2Bub in a transcription-free, reconstituted invitro system. Taken together, our results argue for an active role for Paf1C in promoting H2Bub and ensuring its proper localization invivo.

Directed Differentiation of Zebrafish Pluripotent Embryonic Cells to Functional Cardiomyocytes.

SummaryA cardiomyocyte differentiation in vitro system from zebrafish embryos remains to be established. Here, we have determined pluripotency window of zebrafish embryos by analyzing their gene-expression patterns of pluripotency factors together with markers of three germ layers, and have found that zebrafish undergoes a very narrow period of pluripotency maintenance from zygotic genome activation to a brief moment after oblong stage. Based on the pluripotency and a combination of appropriate conditions, we established a rapid and efficient method for cardiomyocyte generation in vitro from primary embryonic cells. The induced cardiomyocytes differentiated into functional and specific cardiomyocyte subtypes. Notably, these in vitro generated cardiomyocytes exhibited typical contractile kinetics and electrophysiological features. The system provides a new paradigm of cardiomyocyte differentiation from primary embryonic cells in zebrafish. The technology provides a new platform for the study of heart development and regeneration, in addition to drug discovery, disease modeling, and assessment of cardiotoxic agents.

Midbrain-like Organoids from Human Pluripotent Stem Cells Contain Functional Dopaminergic and Neuromelanin-Producing Neurons

Recent advances in 3D culture systems have led to the generation of brain organoids that resemble different human brain regions; however, a 3D organoid model of the midbrain containing functional midbrain dopaminergic (mDA) neurons has not been reported. We developed a method to differentiate human pluripotent stem cells into a large multicellular organoid-like structure that contains distinct layers of neuronal cells expressing characteristic markers of human midbrain. Importantly, we detected electrically active and functionally mature mDA neurons and dopamine production in our 3D midbrain-like organoids (MLOs). In contrast to human mDA neurons generated using 2D methods or MLOs generated from mouse embryonic stem cells, our human MLOs produced neuromelanin-like granules that were structurally similar to those isolated from human substantia nigra tissues. Thus our MLOs bearing features of the human midbrain may provide a tractable invitro system to study the human midbrain and its related diseases.

Ruminal phytate degradation of maize grain and rapeseed meal invitro and as affected by phytate content in donor animal diets and inorganic phosphorus in the buffer.

The ruminal disappearance of phytate phosphorus (InsP6 -P) from maize grain and rapeseed meal (RSM) was determined in two invitro studies. In experiment 1, two diets differing in phosphorus (P) and InsP6 -P concentration were fed to the donor animals of rumen fluid (diet HP: 0.49% P in dry matter, diet LP: 0.29% P). Maize grain and RSM were incubated in a rumen fluid/saliva mixture for 3, 6, 12 and 24h. In experiment 2, a diet similar to diet HP was fed, and the rumen fluid was mixed with artificial saliva containing 120mg inorganic P/l (Pi) or no inorganic P (P0). Maize grain and RSM were incubated with either buffer for 3, 6, 12 and 24h. Total P (tP) and InsP6 concentration were analysed in the fermenter fluids and feed residues. The disappearance of InsP6 -P from maize was completed after 12h of incubation in both experiments. From RSM, 93% (diet LP) and 99% (diet HP) of the InsP6 -P in experiment 1 and 80% (Pi) and 89% (P0) in experiment 2 had disappeared after 24h of incubation. InsP6 -P disappearance was higher when diet HP was fed (maize: 3 and 6h; RSM: 6 and 24h of incubation) and when rumen fluid was mixed with buffer P0 (maize: 6h; RSM: 12 and 24h of incubation). InsP6 -P concentration in the fermenter fluids was higher for maize, but no accumulation of InsP6 -P occurred, indicating a prompt degradation of soluble InsP6 . These results confirmed the capability of rumen micro-organisms to efficiently degrade InsP6 . However, differences between the feedstuffs and diet composition as well as the presence of inorganic P in the invitro system influenced the degradation process. Further studies are required to understand how these factors affect InsP6 degradation and their respective relevance invivo.

Cyclic tensile stress of human annulus fibrosus cells induces MAPK activation: involvement in proinflammatory gene expression

To study the role of mitogen-activated protein kinases (MAPKs) in human annulus fibrosus (AF) cells subjected to cyclic tensile stress (CTS). An invitro system for CTS studies was established using AF cultures on fibronectin-coated silicone dishes. MAPK phosphorylation was studied by western analysis, while gene expression was followed by qRT-PCR. DNA synthesis was assessed by both tritiated thymidine incorporation and flow cytometry, and collagen synthesis using tritiated proline incorporation and the protease-free collagenase method. All three MAPKs studied, i.e., ERK, SAPK/JNK, and p38 were found to be phosphorylated immediately after CTS application within physiological range. A second wave of phosphorylation appeared at later time points. MAPK activation was elevated at higher CTS magnitudes, but independent of the frequency. CTS did not stimulate DNA synthesis neither extracellular matrix turnover, but it stimulated the proinflammatory genes, COX-2, IL-6, and IL-8. This stimulation was more intense at the highest magnitude (8%) testedand at the median frequency (1Hz) and time interval (12h). Blocking of ERK, SAPK/JNK, and p38 MAPK inhibited the CTS-induced stimulation of COX-2 and IL-8, while IL-6 expression was mediated only by SAPK/JNK and p38 MAPK. We have described for the first time the activation of MAPKs in human AF cells in response to CTSand showed that it drives an inflammatory reaction. These observations shed light on the mechanisms of intervertebral disc (IVD) cell responses to mechanical stress, contributing to the understanding of disc pathophysiologyand possibly to the design of novel therapeutic interventions.

Coptidis Rhizoma: protective effects against peroxynitrite-induced oxidative damage and elucidation of its active components

We have investigated the protective effects of Coptidis Rhizoma against peroxynitrite (ONOO(-))-induced oxidative damage and have elucidated the active components of this preparation. In an in-vitro system, Coptidis Rhizoma extract scavenged ONOO(-) and its precursors, nitric oxide (NO) and superoxide anion (O(2)(-)). This scavenging activity was more marked for ONOO(-) than its precursors. In addition, against 3-morpholinosydnonimine-induced cellular damage, this extract significantly reduced cellular ONOO(-) formation and increased cell viability. In an in-vivo lipopolysaccharide plus ischaemia-reperfusion system that generated ONOO(-), the administration of Coptidis Rhizoma extract at 50 and 100 mg kg(-1)/day for 30 days exerted greater inhibition of ONOO(-) than NO and O(2)(-). This suggested that it acted as a direct scavenger of ONOO(-) rather than as a scavenger of its precursors. Moreover, the suppression of the activities of the antioxidative enzymes superoxide dismutase, catalase and glutathione peroxidase was significantly attenuated by the administration of Coptidis Rhizoma extract. Furthermore, the extract ameliorated renal dysfunction judged by decreasing serum urea nitrogen and creatinine levels. To elucidate the active components of Coptidis Rhizoma extract, we evaluated and compared the effects of the phenol plus alkaloid and alkaloid fractions on ONOO-induced damage. We found that the alkaloid fraction consisting of berberine, palmatine and coptisine was the most effective at protecting against ONOO(-). We confirmed that berberine (10 and 20 mg kg(-1)/day for 10 days), the main and most active alkaloid in Coptidis Rhizoma extract, was also protective, exerting NO-, O(2)(-)- and ONOO(-)-scavenging activities. This study suggested that Coptidis Rhizoma could protect against ONOO(-)-induced oxidative damage and that this effect was mainly attributable to the constituent alkaloids, especially berberine. This study is the first to demonstrate an antioxidative effect of alkaloids, including berberine, against ONOO(-)-induced damage.