Dexamethasone is a regulator of clock genes in testicular peritubular cells.

Background: We recently found that peritubular cells of the human testis are a dominant site of expression of the glucocorticoid receptor (GR; encoded by NR3C1). Activation of GR by dexamethasone (Dex) strongly influences the phenotype of cultured human testicular peritubular cells (HTPCs), causing massive changes of their proteome and secretome. As glucocorticoids (GC) are also known to set the internal clock of peripheral organs by regulating clock genes, we tested such an influence of Dex in HTPCs.

The Efficiency of In Vitro Differentiation of Primate iPSCs into Cardiomyocytes Depending on Their Cell Seeding Density and Cell Line Specificity.

A thorough characterization of induced pluripotent stem cells (iPSCs) used with in vitro models or therapeutics is essential. Even iPSCs derived from a single donor can exhibit variability within and between cell lines, which can lead to heterogeneity in results and hinder the promising future of cell replacement therapies. In this study, the cell seeding density of human and rhesus monkey iPSCs was tested to maximize the cell line-specific yield of the generated cardiomyocytes.

Follicular reconstruction and neo-oogenesis in xenotransplantation of human ovarian isolated cells derived from chemotherapy-induced POF patients.

Background: Developing new strategies to restore fertility in patients with chemotherapy-induced Premature Ovarian Failure (Chemo-POF) is important. We aimed to construct an Artificial Ovary (AO) by seeding Human Ovarian Cortical Cells (HOCCs) into Human ovarian Decellularized Cortical Tissue (DCT). We assessed the AO's ability to produce new ovarian follicles following xenotransplantation to NMRI mice.

A piggyBac-based platform for genome editing and clonal rhesus macaque iPSC line derivation.

Non-human primates (NHPs) are, due to their close phylogenetic relationship to humans, excellent animal models to study clinically relevant mutations. However, the toolbox for the genetic modification of NHPs is less developed than those for other species like mice. Therefore, it is necessary to further develop and refine genome editing approaches in NHPs.

Generation of Marmoset Monkey iPSCs with Self-Replicating VEE-mRNAs in Feeder-Free Conditions.

The generation and culture of transgene-free induced pluripotent stem cells (iPSCs) from the common marmoset (Callithrix jacchus) present unique challenges due to the fact that the protocols developed for culture of human or mouse pluripotent cells are not sufficiently optimized for this particular monkey species. Here, we describe the procedures for the reprogramming of marmoset fetal fibroblasts to pluripotency with self-replicating mRNAs using a two-step approach, where intermediate primary colonies generated in the first reprogramming step are converted in the second step to iPSCs with customized marmoset culture medium. The resulting iPSCs are free of transgenes and can be maintained in long-term culture in feeder-free conditions.

Controlling the Switch from Neurogenesis to Pluripotency during Marmoset Monkey Somatic Cell Reprogramming with Self-Replicating mRNAs and Small Molecules.

Induced pluripotent stem cells (iPSCs) hold enormous potential for the development of cell-based therapies; however, the safety and efficacy of potential iPSC-based treatments need to be verified in relevant animal disease models before their application in the clinic. Here, we report the derivation of iPSCs from common marmoset monkeys () using self-replicating mRNA vectors based on the Venezuelan equine encephalitis virus (VEE-mRNAs). By transfection of marmoset fibroblasts with VEE-mRNAs carrying the human , and and culture in the presence of small molecule inhibitors CHIR99021 and SB431542, we first established intermediate primary colonies with neural progenitor-like properties.

A translational cellular model for the study of peritubular cells of the testis.

Testicular peritubular cells (TPCs) are smooth muscle-like cells, which form a compartment surrounding the seminiferous tubules. Previous studies employing isolated human testicular peritubular cells (HTPCs) indicated that their roles in the testis go beyond sperm transport and include paracrine and immunological contributions. Peritubular cells from a non-human primate (MKTPCs), the common marmoset monkey, Callithrix jacchus, share a high degree of homology with HTPCs.

A Fosmid-Based System for the Generation of Recombinant Cercopithecine Alphaherpesvirus 2 Encoding Reporter Genes.

The transmission of Macacine alphaherpesvirus 1 (McHV-1) from macaques, the natural host, to humans causes encephalitis. In contrast, human infection with Cercopithecine alphaherpesvirus 2 (CeHV-2), a closely related alphaherpesvirus from African vervet monkeys and baboons, has not been reported and it is believed that CeHV-2 is apathogenic in humans. The reasons for the differential neurovirulence of McHV-1 and CeHV-2 have not been explored on a molecular level, in part due to the absence of systems for the production of recombinant viruses.

Establishment of porcine and human expanded potential stem cells.

We recently derived mouse expanded potential stem cells (EPSCs) from individual blastomeres by inhibiting the critical molecular pathways that predispose their differentiation. EPSCs had enriched molecular signatures of blastomeres and possessed developmental potency for all embryonic and extra-embryonic cell lineages. Here, we report the derivation of porcine EPSCs, which express key pluripotency genes, are genetically stable, permit genome editing, differentiate to derivatives of the three germ layers in chimeras and produce primordial germ cell-like cells in vitro.

Intraoperative loading of calcium phosphate-coated implants with gentamicin prevents experimental Staphylococcus aureus infection in vivo.

Orthopedic device-related infection (ODRI) is a potentially devastating complication arising from the colonization of the device with bacteria, such as Staphylococcus aureus. The aim of this study was to determine if intraoperative loading of a clinically approved calcium phosphate (CaP) coating with gentamicin can protect from ODRI in vivo. First, CaP-coated titanium aluminium niobium (TAN) discs were used to investigate the adsorption and release kinetics of gentamicin in vitro.

Comparative gene expression profiling of pig-derived iPSC-like cells: Effects of induced pluripotency on expression of porcine endogenous retrovirus (PERV).

Background: Porcine induced pluripotent stem cells (piPSCs) offer an alternative strategy in xenotransplantation (XTx). As human endogenous retroviruses (HERV), particularly HERV-K, are highly expressed in natural human stem cells, we compared the expression of porcine endogenous retroviruses (PERV) and retrotransposon LINE-1 (L1) open reading frames 1 and 2 (pORF1 and pORF2) in different piPSC-like cell lines with their progenitors (porcine fetal fibroblasts, pFF).

Methods: Cells reprogrammed via Sleeping Beauty-transposed transcription factors were cultured and analyzed on a custom-designed microarray representing the reference pig genome.

Immortalization of common marmoset monkey fibroblasts by piggyBac transposition of hTERT.

Following a certain type-specific number of mitotic divisions, terminally differentiated cells undergo proliferative senescence, thwarting efforts to expand different cell populations in vitro for the needs of scientific research or medical therapies. The primary cause of this phenomenon is the progressive shortening of the telomeres and the subsequent activation of cell cycle control pathways leading to a block of cell proliferation. Restoration of telomere length by transgenic expression of telomerase reverse transcriptase (TERT) usually results in bypassing of the replicative senescence and ultimately in cell immortalization.

Pigs expressing the human inhibitory ligand PD-L1 (CD 274) provide a new source of xenogeneic cells and tissues with low immunogenic properties.

Background: The programmed cell death-1 (PD-1, CD279)/PD-Ligand1 (PD-L1, CD274) receptor system is crucial for controlling the balance between immune activation and induction of tolerance via generation of inhibitory signals. Expression of PD-L1 is associated with reduced immunogenicity and renders cells and tissues to an immune-privileged/tolerogenic state.

Methods: To apply this concept for clinical xenotransplantation, we generated human (h)PD-L1 transgenic pigs and characterized expression and biological function of the transgene at the cellular level.

Mouse iPSC generated with porcine reprogramming factors as a model for studying the effects of non-silenced heterologous transgenes on pluripotency.

Mouse somatic cells can be reprogrammed to pluripotency by the ectopic expression of four pluripotency transcription factors, Oct4, Sox2, cmyc, and Klf4. Usually, silencing of the exogenous reprogramming factors is considered to be essential for complete reprogramming and differentiation. In the vast majority of studies, murine pluripotency transcription factor sequences have been used for the reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPSC).

Evaluation of porcine stem cell competence for somatic cell nuclear transfer and production of cloned animals.

Porcine somatic cell nuclear transfer (SCNT) has been used extensively to create genetically modified pigs, but the efficiency of the methodology is still low. It has been hypothesized that pluripotent or multipotent stem cells might result in increased SCNT efficacy as these cells are closer than somatic cells to the epigenetic state found in the blastomeres and therefore need less reprogramming. Our group has worked with porcine SCNT during the last 20 years and here we describe our experience with SCNT of 3 different stem cell lines.

Systematic in vitro and in vivo characterization of Leukemia-inhibiting factor- and Fibroblast growth factor-derived porcine induced pluripotent stem cells.

Derivation and stable maintenance of porcine induced pluripotent stem cells (piPSCs) is challenging. We herein systematically analyzed two piPSC lines, derived by lentiviral transduction and cultured under either leukemia inhibitory factor (LIF) or fibroblast growth factor (FGF) conditions, to shed more light on the underlying biological mechanisms of porcine pluripotency. LIF-derived piPSCs were more successful than their FGF-derived counterparts in the generation of in vitro chimeras and in teratoma formation.

Kidneys From α1,3-Galactosyltransferase Knockout/Human Heme Oxygenase-1/Human A20 Transgenic Pigs Are Protected From Rejection During Ex Vivo Perfusion With Human Blood.

Unlabelled: Multiple modifications of the porcine genome are required to prevent rejection after pig-to-primate xenotransplantation. Here, we produced pigs with a knockout of the α1,3-galactosyltransferase gene (GGTA1-KO) combined with transgenic expression of the human anti-apoptotic/anti-inflammatory molecules heme oxygenase-1 and A20, and investigated their xenoprotective properties.

Methods: The GGTA1-KO/human heme oxygenase-1 (hHO-1)/human A20 (hA20) transgenic pigs were produced in a stepwise approach using zinc finger nuclease vectors targeting the GGTA1 gene and a Sleeping Beauty vector coding for hA20.

Long-Term Culture of Porcine Induced Pluripotent Stem-Like Cells Under Feeder-Free Conditions in the Presence of Histone Deacetylase Inhibitors.

The reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) is a complex process that involves significant epigenetic alterations in the reprogrammed cells. Epigenetic modifiers such as histone deacetylase (HDAC) inhibitors have been shown to increase the efficiency of derivation of iPSCs in humans and mice. In this study, we used three HDAC inhibitors, valproic acid, sodium butyrate, and suberoylanilide hydroxamic acid, together with ascorbic acid, for derivation and long-term feeder-free culture of porcine iPS-like cells.

The small molecule inhibitors PD0325091 and CHIR99021 reduce expression of pluripotency-related genes in putative porcine induced pluripotent stem cells.

Small molecule inhibitors of the mitogen-activated protein kinase kinase (MEK) and glycogen synthesis kinase 3 (Gsk3) have been essential in the establishment and maintenance of embryonic stem cells (ESCs) from rats and from nonpermissive mouse strains. However, conflicting results have been reported regarding their efficacy in the establishment and maintenance of pluripotent stem cells from other species. Here, we investigated the effects of PD0325091 (PD; a MEK inhibitor) and CHIR99021 (CH; a Gsk3β inhibitor) on the reprogramming of porcine fetal fibroblasts to induced pluripotent stem cells (piPSCs).

Passage number of porcine embryonic germ cells affects epigenetic status and blastocyst rate following somatic cell nuclear transfer.

Epigenetic instability of donor cells due to long-term in vitro culture may influence the success rate of subsequent somatic cell nuclear transfer (SCNT). Therefore, the present study was designed (1) to investigate the epigenetic changes after prolonged culture in vitro of porcine embryonic germ (EG) cells, including differences in expression levels of both DNA methylation and demethylation-related genes and catalyses of histone modifications, and (2) to assess the efficiency of SCNT using EG cells from different passages. Results showed that genes either associated with DNA demethylation including DNMTs and TET1 or genes related to histone acetylation including HDACs were highly expressed in EG cells at higher passages when compared to EG cells at lower passages.

The choice of expression vector promoter is an important factor in the reprogramming of porcine fibroblasts into induced pluripotent cells.

Porcine induced pluripotent stem cells (iPSCs) are an important animal model for development of regenerative therapies in human medicine. To date, the majority of the porcine cell lines with iPSC characteristics have been generated with the use of viral vectors harboring human or mouse reprogramming factors. Here, we report on the use of Sleeping Beauty transposon vectors based on the porcine transcription factor sequences to reprogram porcine fetal fibroblasts into iPSC-like cells.

In vitro culture and characterization of putative porcine embryonic germ cells derived from domestic breeds and Yucatan mini pig embryos at Days 20-24 of gestation.

Embryonic germ cells (EGC) are cultured pluripotent cells derived from primordial germ cells (PGC). This study explored the possibility of establishing porcine EGC from domestic breeds and Yucatan mini pigs using embryos at Days 17-24 of gestation. In vitro culture of PGC from both pooled and individual embryos resulted in the successful derivation of putative EGC lines from Days 20 to 24 with high efficiency.

Methylation changes in porcine primordial germ cells.

Epigenetic re-programming is an important event in the development of primordial germ cells (PGC) into functional gametes, characterized by genome-wide erasure of DNA methylation and re-establishment of epigenetic marks, a process essential for restoration of the potential for totipotency. In this study changes in the methylation status of centromeric repeats and two IGF2-H19 differentially methylated domain (DMD) sequences were examined in porcine PGC between Days 24 and 31 of pregnancy. The methylation levels of centromeric repeats and IGF2-H19 DMD sequences decreased rapidly from Days 24 to 28 in both male and female PGC.