The Effect of Marijuana on Postoperative Spine Patients' Emergency Department Visits, Readmission Rates, and Opioid Consumption.

OBJECTIVE: To compare postoperative opioid consumption with patients who tested negative for tetrahydrocannabinol (THC) preoperatively with those who were THC-positive and patients who were positive for THC and any other drug and to compare 90-day rates of postoperative emergency department (ED) visits and 90-day readmission rates, using morphine milligram equivalents (MMEs), for those three patient populations. METHODS: Three patient groups were confirmed with preoperative urine drug screens. Chart reviews were conducted to determine whether there was an ED visit or hospital readmission 90 days from the index procedure. MMEs were calculated for all patients. RESULTS: There were a total of 252 patients in the THC-negative control group, 54 in the THC-positive group, and 47 in the THC-and-opioid-positive group. The 90-day ED visit and 90-day readmission rates were not statistically significant among the groups. Both the multidrug and THC-only-positive patients showed a higher 90-day MME compared with the control patients. DISCUSSION: Our study demonstrates that THC used may increase opioid consumption. The THC patients to be cautious toward are the multidrug user. Although not statistically significant, multidrug patients were noted for a trend toward increased ED visits and readmissions.

Optimization of mTOR Inhibitors Using Property-Based Drug Design and Free-Wilson Analysis for Improved In Vivo Efficacy.

The mTOR kinase regulates a variety of critical cellular processes and has become a target for the treatment of various cancers. Using a combination of property-based drug design and Free-Wilson analysis, we further optimized a series of selective mTOR inhibitors based on the (S)-6a-methyl-6a,7,9,10-tetrahydro[1,4]oxazino[3,4-h]pteridin-6(5H)-one scaffold. Our efforts resulted in 14c, which showed similar in vivo efficacy compared to previous lead 1 at 1/15 the dose, a result of its improved drug-like properties.

Effect of foot stato-dynamic disorders on hemodynamics of the lower limb using strain-gauge plethysmography.

BACKGROUND: The plantar venous pump (PVP), composed of deep plantar veins, is the most distal contributor to venous return from the lower limbs. A pressing need still exists to assess how plantar muscle contraction and gait affect PVP function, how foot stato-dynamic disorders (FSDs) can contribute to venous insufficiency, and how venous return can be optimally stimulated. Our first objective is to compare the venous blood hemodynamics in lower limbs between healthy subjects with a FSD and healthy subjects without a FSD to understand the influence of foot morphology in the performance of the PVP. Our second objective is to evaluate whether PVP function varies with different plantar pressures. METHODS: A total of 52 healthy volunteers (26 feet with a normal arch as the control group and 26 feet with dysmorphism [13 flat feet and 13 hollow feet]) were included. Strain-gauge plethysmography was performed 8 cm above the medial malleolus at different conditions of PVP stimulation: (1) toe flexion, (2) intermittent pneumatic compression (IPC) with and without an insole, and (3) 3-km/h speed walking on a treadmill barefoot, with shoes, and with shoes and insoles. From the strain-gauge plethysmography, we measured the venous blood ejection fraction (EF). From the pressure sensor placed at the midfoot on the plantar arch during IPC, we obtained the maximal pressure (N/cm2). RESULTS: Toe flexion allowed for ejection of an average of 20% of the total venous volume in both groups. IPC and gait generated a mean EF superior to 100% of the available venous volume. The maximal pressure applied at the midfoot during IPC was lower than the pressure set. No significant differences in the EF or maximal pressure were observed between the two groups. The mean EF was not significantly affected for the pronator and supinator walkers compared with those with normal walking dynamics. Wearing shoes did not significantly affect the mean EF. However, wearing insoles during gait significantly increased the venous return in feet with plantar dysmorphism. CONCLUSIONS: To the best of our knowledge, this clinical study is the first to assess the PVP function in 52 healthy volunteers with and without FSDs. We found that wearing shoes did not significantly affect PVP efficiency but that wearing morphologically adapted insoles significantly improved the venous return in the dysmorphic feet. In our sample of healthy volunteers, the differences observed between the control group and feet with FSDs were not statistically significant.

Effect of dysmorphic plantar arch on venous hemodynamics of the lower limb.

BACKGROUND: The role of the plantar venous pump (PVP) on venous return is evident but the effects of the foot morphology have never been characterized properly. METHOD: 52 healthy volunteers-26 with normal plantar arch (control) and 26 with dysmorphic plantar arch (in two subgroups: 13 flat feet, 13 hollow feet)-were included. Using Doppler ultrasound, we measured the diameter and the peak systolic velocity in the large veins of the lower limb after PVP stimulation by manual compression and bodyweight transfer. RESULT: The mean peak systolic velocity of the studied veins varied from 12.2 cm/s to 41.7 cm/s in the control group and from 10.9 cm/s to 39.1 cm/s in the dysmorphic plantar group. The foot arch morphology did not affect significantly the venous blood flows, except in the great saphenous vein during manual compression. CONCLUSION: The plantar morphology did not induce a significant increase of venous blood velocity resulting from PVP stimulation.

Machine learning and hypothesis driven optimization of bull semen cryopreservation media.

Cryopreservation provides a critical tool for dairy herd genetics management. Due to widely varying inter- and within-bull post thaw fertility, recent research on cryoprotectant extender medium has not dramatically improved suboptimal post-thaw recovery in industry. This progress is stymied by the interactions between samples and the many components of extender media and is often compounded by industry irrelevant sample sizes. To address these challenges, here we demonstrate blank-slate optimization of bull sperm cryopreservation media by supervised machine learning. We considered two supervised learning models: artificial neural networks and Gaussian process regression (GPR). Eleven media components and initial concentrations were identified from publications in bull semen cryopreservation, and an initial 200 extender-post-thaw motility pairs were used to train and 32 extender-post-thaw motility pairs to test the machine learning algorithms. The median post-thaw motility after coupling differential evolution with GPR the increased from 52.6 ± 6.9% to 68.3 ± 6.0% at generations 7 and 17 respectively, with several media performing dramatically better than control media counterparts. This is the first study in which machine learning was used to determine the best combination of constituents to optimize bull sperm cryopreservation media, and provides a template for optimization in other cell types.

Hydroxytyrosol and resveratrol improves kinetic and flow cytometric parameters of cryopreserved bull semen with low cryotolerance.

There is considerable interest in breeding programs to "rescue" semen with poor post-thaw fertility from bulls known as "bad freezers". We hypothesized that there may be an interaction between the post-thaw recovery of sperm and the efficacy of antioxidant addition to extenders. The current study assesses the effects of antioxidant additives hydroxytyrosol (HT) and resveratrol (RSV) on the post-thaw semen parameters in two groups of bulls classified as either high or low cryotolerant (i.e., "good" and "bad" freezers). Semen samples were collected from 40 bulls and processed in the extenders containing different concentrations of HT (experiment 1; 0, 25 and 50 µM) and RSV (experiment 2; 0.0, 0.01, 0.1 and 1 mM). In experiment 1, bulls in the low cryotolerance group had a significant improvement in post-thaw recovery at 25 µM and 50 µM (P < 0.05). These improvements were observed in motility and several cellular parameters. However, post-thaw semen quality in the high cryotolerance group was not significantly affected by the HT addition. In experiment 2, although RSV did not have any positive impact in high cryotolerance group, post-thaw recovery in the low cryotolerance bulls was significantly improved in 0.1 mM RSV. Oxidative stress markers in either high or low cryotolerance groups were not significantly changed by RSV addition (P > 0.05). It can be concluded that addition of optimized concentrations of HT and RSV to the extender could be a strategy for improving the post-thaw semen, especially in bulls with high genetic merit but low initial cryotolerance.

Effectiveness of Corticosteroid Injections in Diabetic Patients With De Quervain Tenosynovitis.

PURPOSE: We sought to determine the effectiveness of corticosteroid injections (CSIs) for de Quervain tenosynovitis in patients with diabetes mellitus. METHODS: We retrospectively identified all patients with diabetes receiving a CSI for de Quervain tenosynovitis by 16 surgeons over a 2-year period. Data collected included demographic information, medical comorbidities, number and timing of CSIs, and first dorsal compartment release. Success was defined as not undergoing an additional CSI or surgical intervention. The mixture of a corticosteroid and local anesthetic provided in each injection was at the discretion of each individual surgeon. RESULTS: Corticosteroid injections were given to 169 wrists in 169 patients with diabetes. Out of 169 patients, 83 (49%) had success following the initial CSI, 44 (66%) following a second CSI, and 6 (67%) following a third CSI. A statistically significant difference was identified in the success rates between the first and second CSIs. Ultimately, 36 of 169 wrists (21%) underwent a first dorsal compartment release. CONCLUSIONS: Patients with diabetes mellitus have a decreased probability of success following a single CSI for de Quervain tenosynovitis in comparison to nondiabetic patients, as described in the literature. However, the effectiveness of each additional CSI does not appear to diminish. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic IV.

[SARS-CoV-2 and Perinatal Aspects]. / Perinatale Aspekte der SARS-CoV-2 Infektion.

The novel coronavirus SARS-CoV-2 has developed into a pandemic, yet still has many unknowns. The modalities of transmission, different symptoms and manifestations as well as concomitant circumstances of the disease are insufficiently characterized. Especially patient groups in special situations like pregnant women and newborns have to be considered separately. The current knowledge about pregnancy, labor and the first days of life is characterized by particular uncertainty due to the scarce data available. However, there is currently no evidence of significant unfavorable maternal and perinatal outcome. Many pregnant women with SARS-CoV-2 infection remain asymptomatic. The possibility of vertical transmission to the child cannot be excluded with certainty. However, indications of vertical transmission were detected only in individual cases. Newborn infections are also rather rare, unspecific and usually mild, with respiratory symptoms dominating. In this article, the data available to date are examined in order to provide better information, advice and treatment for pregnant women and newborns with SARS-CoV-2 and to provide suggestions for future research.

Low concentrations of 3-O-methylglucose improve post thaw recovery in cryopreserved bovine spermatozoa.

A number of studies have explored the use of membrane permeable (usually metabolizable) and membrane impermeable saccharides to protect cells in general, and sperm in particular during cryopreservation. Critical concentrations for protective levels of sugars frequently range between 50 mmol/L and 500 mmol/L, where efficacy is attributed to the sugar's membrane stabilizing and glass forming attributes and colligative effects that reduce intra- and extracellular salt concentrations during freezing. Many studies on bull sperm have demonstrated that both permeating and non-permeating sugars have negligible positive effects on post-thaw viability. Recently, however, a non-metabolizable sugar, 3-O-Methylglucose (3-OMG), was shown to protect hepatocytes during liver cryopreservation at 0.1-0.3 mol/L. Because glucose is readily transported into sperm, we hypothesized that 3-OMG could be a new class of cryoprotectant to explore in bull sperm. Here we present positive results demonstrating that 3-OMG improves post thaw viability in bull sperm, and that this effect is not likely due to improved glass forming capabilities. In particular, in experiment 1, 3-OMG was added to the Tris-egg yolk-glycerol base media at levels from 0 mmol/L to 200 mmol/L. Semen from four bulls was collected and diluted with one of the cryopreservation media, cooled, and frozen following industry standard practices. Motility and mitochondrial activity were negatively impacted when concentration of 3-OMG was more than 25 mmol/L. Therefore, we explored lower concentrations in experiment 2, where semen from eight bulls was used to evaluate concentrations 5 mmol/L, 15 mmol/L and 25 mmol/L of 3-OMG compared with control. Motility and progressive motility in 5 mmol/L 3-OMG and in the control were significantly higher than 15 mmol/L and 25 mmol/L 3-OMG, whereas mitochondrial activity and acrosome integrity in 5 mmol/L 3-OMG were significantly better than the control freezing medium. In experiment 3, to evaluate whether the improved effects of 3-OMG are due to its non-metabolizing property, or due to colligative effects, we compared post-thaw viability in semen from four bulls cryopreserved with 5 mmol/L glucose, sucrose, or 3-OMG. Motility and progressive motility was significantly improved in 3-OMG compared to glucose or sucrose groups which were comparable to the EY control. In conclusion, 3-OMG at a concentration of 5 mmol/L in Tris-egg yolk-glycerol medium improves the post thaw motility, progressive motility and viability of bull sperm. The mechanism of action is not understood but because the efficacy is maximal at low concentrations, it is not likely due to improved intra- or extracellular glass forming abilities and may demonstrate a different protective mechanism than was shown in hepatocytes.

Rational discovery of a highly novel and selective mTOR inhibitor.

Aided by Structure Based Drug Discovery (SBDD), we rapidly designed a highly novel and selective series of mTOR inhibitors. This chemotype conveys exquisite kinase selectivity, excellent in vitro and in vivo potencies and ADME safety profiles. These compounds could serve as good tools to explore the potential of TORC inhibition in various human diseases.

Proteomic markers of low and high fertility bovine spermatozoa separated by Percoll gradient.

In the context of artificial insemination, male fertility is defined as the ability to produce functional spermatozoa able to withstand cryopreservation. We hypothesized that interindividual variations in fertility depend on the proportion of the fully functional sperm population contained in the insemination dose. The objective of this study was to identify protein markers of the fully functional sperm subpopulation. Insemination doses from four high-fertility (HF) and four low-fertility (LF) bulls with comparable post-thaw quality parameters were selected for proteomic analysis using iTRAQ technology. Thawed semen was centrifuged through a Percoll gradient to segregate the motile (high density [HD]) from the immotile (low density [LD]) sperm populations. Sperm proteins were extracted with sodium deoxycholate and four groups were compared: LD and HD spermatozoa from LF and HF bulls. A total of 498 unique proteins were identified and quantified. Comparison of HD spermatozoa from HF and LF bulls revealed that five proteins were significantly more abundant in the HF group (AK8, TPI1, TSPAN8, OAT, and DBIL5) whereas five proteins were more abundant in the LF group (RGS22, ATP5J, CLU, LOC616319, and CCT5). Comparison of LD spermatozoa from HF and LF bulls revealed that four proteins were significantly more abundant in the HF group (IL4I1, CYLC2, OAT, and ARMC3) whereas 15 proteins were significantly more abundant in the LF group (HADHA, HSP90AA1, DNASE1L3, SLC25A20, GPX5, TCP1, HIP1, CLU, G5E622, LOC616319, HSPA2, NUP155, DPY19L2, SPERT, and SERPINE2). DBIL5, TSPAN8, and TPI1 showed potential as putative markers of the fully functional sperm subpopulation.

MET Tyrosine Kinase Inhibition Enhances the Antitumor Efficacy of an HGF Antibody.

Receptor tyrosine kinase therapies have proven to be efficacious in specific cancer patient populations; however, a significant limitation of tyrosine kinase inhibitor (TKI) treatment is the emergence of resistance mechanisms leading to a transient, partial, or complete lack of response. Combination therapies using agents with synergistic activity have potential to improve response and reduce acquired resistance. Chemoreagent or TKI treatment can lead to increased expression of hepatocyte growth factor (HGF) and/or MET, and this effect correlates with increased metastasis and poor prognosis. Despite MET's role in resistance and cancer biology, MET TKI monotherapy has yielded disappointing clinical responses. In this study, we describe the biological activity of a selective, oral MET TKI with slow off-rate and its synergistic antitumor effects when combined with an anti-HGF antibody. We evaluated the combined action of simultaneously neutralizing HGF ligand and inhibiting MET kinase activity in two cancer xenograft models that exhibit autocrine HGF/MET activation. The combination therapy results in additive antitumor activity in KP4 pancreatic tumors and synergistic activity in U-87MG glioblastoma tumors. Pharmacodynamic characterization of biomarkers that correlate with combination synergy reveal that monotherapies induce an increase in the total MET protein, whereas combination therapy significantly reduces total MET protein levels and phosphorylation of 4E-BP1. These results hold promise that dual targeting of HGF and MET by combining extracellular ligand inhibitors with intracellular MET TKIs could be an effective intervention strategy for cancer patients who have acquired resistance that is dependent on total MET protein. Mol Cancer Ther; 16(7); 1269-78. ©2017 AACR.

Evaluation of the therapeutic efficacy of a MEK inhibitor (TAK-733) using ¹8F-fluorodeoxyglucose-positron emission tomography in the human lung xenograft model A549.

OBJECTIVE: The aim of this study was to evaluate the potential of (18)F-fluorodeoxyglucose-positron emission tomography ((18)F-FDG-PET) for monitoring the therapeutic efficacy of TAK-733, an inhibitor of mitogen-activated protein kinase kinase, in nude rats bearing A549 (human lung carcinoma) xenografts. METHODS: TAK-733 was administered orally by gavage to nude xenograft rats for 2 weeks, at dosage levels of 0 (0.5% w/v methylcellulose solution), 1, 3, and 10 mg/kg/day (n = 8/dose). Tumor size was measured before treatment (day 0), and on days 1, 3, 7, 9, 11, and 14. PET scans were performed pretreatment (day 0), and on days 2, 4, 7, 10, and 14. Tracer accumulations in tumor tissue were quantified as the mean standard uptake value (SUVmean). RESULTS: No deaths or treatment-related body weight losses occurred during the study period. TAK-733 showed dose-dependent inhibition of tumor growth and (18)F-FDG uptake in tumor tissue. At a dosage of 10 mg/kg, TAK-733 treatment produced a statistically significant reduction in tumor weight from day 11 compared with the vehicle group (P < 0.05). Tumor growth was inhibited in the 10 mg/kg group with a treated/control value of 31% on day 14. The SUVmean on day 2 in this dosage group was statistically lower than that observed on day 0, and that seen in the vehicle group on day 2 (P < 0.05 for both comparisons). Furthermore, this reduction in SUVmean at 10 mg/kg was maintained over time. In the two lower dosage groups (1 and 3 mg/kg), SUVmean gradually increased over time. CONCLUSIONS: (18)F-FDG-PET enabled early determination of late anti-tumor activity in response to TAK-733 treatment.

Antitumor activity of the MEK inhibitor TAK-733 against melanoma cell lines and patient-derived tumor explants.

The goal of this study was to investigate the activity of the selective MEK1/2 inhibitor TAK-733 in both melanoma cell lines and patient-derived melanoma xenograft models. In vitro cell proliferation assays using the sulforhodamine B assay were conducted to determine TAK-733 potency and melanoma responsiveness. In vivo murine modeling with eleven patient-derived melanoma explants evaluated daily dosing of TAK-733 at 25 or 10 mg/kg. Immunoblotting was performed to evaluate on-target activity and downstream inhibition by TAK-733 in both in vitro and in vivo studies. TAK-733 demonstrated broad activity in most melanoma cell lines with relative resistance observed at IC50 > 0.1 µmol/L in vitro. TAK-733 also exhibited activity in 10 out of 11 patient-derived explants with tumor growth inhibition ranging from 0% to 100% (P < 0.001-0.03). Interestingly, BRAF(V600E) and NRAS mutational status did not correlate with responsiveness to TAK-733. Pharmacodynamically, pERK was suppressed in sensitive cell lines and tumor explants, confirming TAK-733-mediated inhibition of MEK1/2, although the demonstration of similar effects in the relatively resistant cell lines and tumor explants suggests that escape pathways are contributing to melanoma survival and proliferation. These data demonstrate that TAK-733 exhibits robust tumor growth inhibition and regression against human melanoma cell lines and patient-derived xenograft models, suggesting that further clinical development in melanoma is of scientific interest. Particularly interesting is the activity in BRAF wild-type models, where current approved therapy such as vemurafenib has been reported not to be active.

Oxytocin improves the expression of cardiac specific markers in porcine bone marrow stem cells differentiation.

Bone marrow stem cells (BMSCs) treated with 5-azacytidine possess myogenic differentiation potential. Oxytocin (OT) induces cardiomyogenesis in murine embryonic and cardiac stem cells. We attempted to isolate, characterize, and induce OT-mediated cardiomyogenic differentiation of porcine pBMSCs. Cells were treated as: control, OT, and 5-azacytidine groups. During early passages, transcripts of Oct4, GATA4, OT receptor, and phospholamban were expressed. RT-PCR showed upregulation of GATA4 in OT and 5-azacytidine-induced groups. Immunocytochemistry revealed higher expressions of cardiac troponin T and myosin heavy chain in OT than in 5-azacytidine-induced groups (p < 0.01). Western blot analysis showed upregulation of cardiac troponin I in OT-induced pBMSCs (p < 0.01). We infer pBMSCs should be induced during early passages, when expressing transcription factors related to pluripotency and cardiomyogenesis, as well as OT receptor. The more abundant expression of cardiac specific proteins in OT-treated pBMSCs suggests OT could be a more potent cardiomyogenic inducer of pBMSC.

Discovery and synthesis of novel 4-aminopyrrolopyrimidine Tie-2 kinase inhibitors for the treatment of solid tumors.

The synthesis and biological evaluation of novel Tie-2 kinase inhibitors are presented. Based on the pyrrolopyrimidine chemotype, several new series are described, including the benzimidazole series by linking a benzimidazole to the C5-position of the 4-amino-pyrrolopyrimidine core and the ketophenyl series synthesized by incorporating a ketophenyl group to the C5-position. Medicinal chemistry efforts led to potent Tie-2 inhibitors. Compound 15, a ketophenyl pyrrolopyrimidine urea analog with improved physicochemical properties, demonstrated favorable in vitro attributes as well as dose responsive and robust oral tumor growth inhibition in animal models.

Biochemical and pharmacological characterization of human c-Met neutralizing monoclonal antibody CE-355621.

The c-Met proto-oncogene is a multifunctional receptor tyrosine kinase that is stimulated by its ligand, hepatocyte growth factor (HGF), to induce cell growth, motility and morphogenesis. Dysregulation of c-Met function, through mutational activation or overexpression, has been observed in many types of cancer and is thought to contribute to tumor growth and metastasis by affecting mitogenesis, invasion, and angiogenesis. We identified human monoclonal antibodies that bind to the extracellular domain of c-Met and inhibit tumor growth by interfering with ligand-dependent c-Met activation. We identified antibodies representing four independent epitope classes that inhibited both ligand binding and ligand-dependent activation of c-Met in A549 cells. In cells, the antibodies antagonized c-Met function by blocking receptor activation and by subsequently inducing downregulation of the receptor, translating to phenotypic effects in soft agar growth and tubular morphogenesis assays. Further characterization of the antibodies in vivo revealed significant inhibition of c-Met activity (≥ 80% lasting for 72-96 h) in excised tumors corresponded to tumor growth inhibition in multiple xenograft tumor models. Several of the antibodies identified inhibited the growth of tumors engineered to overexpress human HGF and human c-Met (S114 NIH 3T3) when grown subcutaneously in athymic mice. Furthermore, lead candidate antibody CE-355621 inhibited the growth of U87MG human glioblastoma and GTL-16 gastric xenografts by up to 98%. The findings support published pre-clinical and clinical data indicating that targeting c-Met with human monoclonal antibodies is a promising therapeutic approach for the treatment of cancer.

Rat blastocyst-derived stem cells are precursors of embryonic and extraembryonic lineages.

Despite recent advances in the derivation of rat embryonic stem cells, clear comprehension of the timing and mechanisms underlying rat early embryo lineage selection is lacking. We have previously shown the in vivo contribution of rat embryonic stem-like cells exclusively to developing extraembryonic tissues. To elucidate possible mechanisms governing the in vitro and in vivo behaviors of these rat blastocyst-derived stem cells, we evaluated their developmental capacity by using several approaches. Molecular marker analysis demonstrated the expression profile of genes characterizing not only pluripotency but also extraembryonic endoderm and trophoblast. In vitro differentiation through embryoid body formation showed in vitro pluripotent capacity through differentiation into derivatives of all three embryonic germ layers. Following either blastocyst injection, diploid or tetraploid aggregation, and embryo transfer, these rat blastocyst-derived stem cells also demonstrated in vivo multipotency through contribution to multiple developmentally distinct extraembryonic lineages. Features of phenotypic heterogeneity were revealed following examination of cell line morphology and culture behavior, as well as quantitative analysis of marker expression in discrete undifferentiated and differentiated populations of cells by flow cytometry. We demonstrate for the first time that stem cells derived from the rat blastocyst have the ability to contribute to the embryonic and extraembryonic lineages. Together, these results provide a valuable new model for rat stem cell biology and for the elucidation of early lineage selection in the embryo.

Induced pluripotent stem cell lines derived from equine fibroblasts.

The domesticated horse represents substantial value for the related sports and recreational fields, and holds enormous potential as a model for a range of medical conditions commonly found in humans. Most notable of these are injuries to muscles, tendons, ligaments and joints. Induced pluripotent stem (iPS) cells have sparked tremendous hopes for future regenerative therapies of conditions that today are not possible to cure. Equine iPS (EiPS) cells, in addition to bringing promises to the veterinary field, open up the opportunity to utilize horses for the validation of stem cell based therapies before moving into the human clinical setting. In this study, we report the generation of iPS cells from equine fibroblasts using a piggyBac (PB) transposon-based method to deliver transgenes containing the reprogramming factors Oct4, Sox2, Klf4 and c-Myc, expressed in a temporally regulated fashion. The established iPS cell lines express hallmark pluripotency markers, display a stable karyotype even during long-term culture, and readily form complex teratomas containing all three embryonic germ layer derived tissues upon in vivo grafting into immunocompromised mice. Our EiPS cell lines hold the promise to enable the development of a whole new range of stem cell-based regenerative therapies in veterinary medicine, as well as aid the development of preclinical models for human applications. EiPS cell could also potentially be used to revive recently extinct or currently threatened equine species.

Trophoblast stem cell marker gene expression in inner cell mass-derived cells from parthenogenetic equine embryos.

Although putative horse embryonic stem (ES)-like cell lines have been obtained recently from in vivo-derived embryos, it is currently not known whether it is possible to obtain ES cell (ESC) lines from somatic cell nuclear transfer (SCNT) and parthenogenetic (PA) embryos. Our aim is to establish culture conditions for the derivation of autologous ESC lines for cell therapy studies in an equine model. Our results indicate that both the use of early-stage blastocysts with a clearly visible inner cell mass (ICM) and the use of pronase to dissect the ICM allow the derivation of a higher proportion of primary ICM outgrowths from PA and SCNT embryos. Primary ICM outgrowths express the molecular markers of pluripotency POU class 5 homeobox 1 (POU5F1) and (sex determining region-Y)-box2 (SOX2), and in some cases, NANOG. Cells obtained after the passages of PA primary ICM outgrowths display alkaline phosphatase (AP) activity and POU5F1, SOX2, caudal-related homeobox-2 (CDX2) and eomesodermin (EOMES) expression, but may lose NANOG. Cystic embryoid body-like structures expressing POU5F1, CDX2 and EOMES were produced from these cells. Immunohistochemical analysis of equine embryos reveals the presence of POU5F1 in trophectoderm, primitive endoderm and ICM. These results suggest that cells obtained after passages of primary ICM outgrowths are positive for trophoblast stem cell markers while expressing POU5F1 and displaying AP activity. Therefore, these cells most likely represent trophoblast cells rather than true ESCs. This study represents an important first step towards the production of autologous equine ESCs for pre-clinical cell therapy studies on large animal models.