An optimized culture system for efficient derivation of porcine expanded potential stem cells from preimplantation embryos and by reprogramming somatic cells.

Pigs share anatomical and physiological traits with humans and can serve as a large-animal model for translational medicine. Bona fide porcine pluripotent stem cells (PSCs) could facilitate testing cell and drug therapies. Agriculture and biotechnology may benefit from the ability to produce immune cells for studying animal infectious diseases and to readily edit the porcine genome in stem cells. Isolating porcine PSCs from preimplantation embryos has been intensively attempted over the past decades. We previously reported the derivation of expanded potential stem cells (EPSCs) from preimplantation embryos and by reprogramming somatic cells of multiple mammalian species, including pigs. Porcine EPSCs (pEPSCs) self-renew indefinitely, differentiate into embryonic and extra-embryonic lineages, and permit precision genome editing. Here we present a highly reproducible experimental procedure and data of an optimized and robust porcine EPSC culture system and its use in deriving new pEPSC lines from preimplantation embryos and reprogrammed somatic cells. No particular expertise is required for the protocols, which take ~4-6 weeks to complete. Importantly, we successfully established pEPSC lines from both in vitro fertilized and somatic cell nuclear transfer-derived embryos. These new pEPSC lines proliferated robustly over long-term passaging and were amenable to both simple indels and precision genome editing, with up to 100% targeting efficiency. The pEPSCs differentiated into embryonic cell lineages in vitro and teratomas in vivo, and into porcine trophoblast stem cells in human trophoblast stem cell medium. We show here that pEPSCs have unique epigenetic features, particularly H3K27me3 levels substantially lower than fibroblasts.

Stem cell-derived porcine macrophages as a new platform for studying host-pathogen interactions.

BACKGROUND: Infectious diseases of farmed and wild animals pose a recurrent threat to food security and human health. The macrophage, a key component of the innate immune system, is the first line of defence against many infectious agents and plays a major role in shaping the adaptive immune response. However, this phagocyte is a target and host for many pathogens. Understanding the molecular basis of interactions between macrophages and pathogens is therefore crucial for the development of effective strategies to combat important infectious diseases. RESULTS: We explored how porcine pluripotent stem cells (PSCs) can provide a limitless in vitro supply of genetically and experimentally tractable macrophages. Porcine PSC-derived macrophages (PSCdMs) exhibited molecular and functional characteristics of ex vivo primary macrophages and were productively infected by pig pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV) and African swine fever virus (ASFV), two of the most economically important and devastating viruses in pig farming. Moreover, porcine PSCdMs were readily amenable to genetic modification by CRISPR/Cas9 gene editing applied either in parental stem cells or directly in the macrophages by lentiviral vector transduction. CONCLUSIONS: We show that porcine PSCdMs exhibit key macrophage characteristics, including infection by a range of commercially relevant pig pathogens. In addition, genetic engineering of PSCs and PSCdMs affords new opportunities for functional analysis of macrophage biology in an important livestock species. PSCs and differentiated derivatives should therefore represent a useful and ethical experimental platform to investigate the genetic and molecular basis of host-pathogen interactions in pigs, and also have wider applications in livestock.

Generating low immunogenic pig pancreatic islet cell clusters for xenotransplantation.

Xenotransplantation of pancreatic islets offers a promising alternative to overcome the shortage of allogeneic donors. Despite significant advances, either immune rejection or oxygen supply in immune protected encapsulated islets remains major bottlenecks for clinical application. To decrease xenogeneic immune responses, we generated tissue engineered swine leucocyte antigen (SLA)-silenced islet cell clusters (ICC). Single-cell suspensions from pancreatic islets were generated by enzymatic digestion of porcine ICCs. Cells were silenced for SLA class I and class II by lentiviral vectors encoding for short hairpin RNAs targeting beta2-microglobulin or class II transactivator, respectively. SLA-silenced ICCs-derived cells were then used to form new ICCs in stirred bioreactors in the presence of collagen VI. SLA class I silencing was designed to reach a level of up to 89% and class II by up to 81% on ICCs-derived cells. Xenogeneic T cell immune responses, NK cell and antibody-mediated cellular-dependent immune responses were significantly decreased in SLA-silenced cells. In stirred bioreactors, tissue engineered islets showed the typical 3D structure and insulin production. These data show the feasibility to generate low immunogenic porcine ICCs after single-cell engineering and post-transduction islet reassembling that might serve as an alternative to allogeneic pancreatic islet cell transplantation.

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. Data were complemented by qRT-PCR and reverse transcriptase (RT) assay. RESULTS: The expression profiles revealed that 8515 of 26 967 targets were differentially expressed. A total of 4443 targets showed log2 expression ratio >1, and 4072 targets showed log2 expression ratio less than -1 with 0.05 P-value threshold. Approximately ten percent of the targets showed highly significant expression ratios with log2 ≥4 or ≤-4. Besides this general switch in cellular gene expression that was accompanied by an altered morphology, expression of both PERV and L1 pORF1/pORF2 was significantly enhanced. piPSC-like cells revealed a 10-fold to 100-fold higher transcription of the viral PERV-A and PERV-B envelope genes (env), viral protease/polymerase (prt/pol), and L1 elements. No functional retrovirus could be detected under these conditions. CONCLUSION: Epigenetic reprogramming has functional impact on retrotransposons. Thus, the induction of pig-derived pluripotent cells influences their PERV expression profile. Data emphasize the necessity to focus on animals, which show non-functional endogenous viral background to ensure virological safety.

Anticonvulsant effects after grafting of rat, porcine, and human mesencephalic neural progenitor cells into the rat subthalamic nucleus.

Cell transplantation based therapy is a promising strategy for treating intractable epilepsies. Inhibition of the subthalamic nucleus (STN) or substantia nigra pars reticulata (SNr) is a powerful experimental approach for remote control of different partial seizure types, when targeting the seizure focus is not amenable. Here, we tested the hypothesis that grafting of embryonic/fetal neural precursor cells (NPCs) from various species (rat, human, pig) into STN or SNr of adult rats induces anticonvulsant effects. To rationally refine this approach, we included NPCs derived from the medial ganglionic eminence (MGE) and ventral mesencephalon (VM), both of which are able to develop a GABAergic phenotype. All VM- and MGE-derived cells showed intense migration behavior after grafting into adult rats, developed characteristics of inhibitory interneurons, and survived at least up to 4 months after transplantation. By using the intravenous pentylenetetrazole (PTZ) seizure threshold test in adult rats, transient anticonvulsant effects were observed after bilateral grafting of NPCs derived from human and porcine VM into STN, but not after SNr injection (site-specificity). In contrast, MGE-derived NPCs did not cause anticonvulsant effects after grafting into STN or SNr (cell-specificity). Neither induction of status epilepticus by lithium-pilocarpine to induce neuronal damage prior to the PTZ test nor pretreatment of MGE cells with retinoic acid and potassium chloride to increase differentiation into GABAergic neurons could enhance anticonvulsant effectiveness of MGE cells. This is the first proof-of-principle study showing anticonvulsant effects by bilateral xenotransplantation of NPCs into the STN. Our study highlights the value of VM-derived NPCs for interneuron-based cell grafting targeting the STN.

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. RESULTS: The hPD-L1 was detected in kidney, heart, and pancreas. In addition, peripheral blood mononuclear cells (PBMC), cultured fibroblasts, and endothelial cells were hPD-L1 positive (hPD-L1+ ). The hPD-L1 levels were increased by the treatment of transgenic cells with human cytokines (eg, TNF-α), suggesting a regulatable mode of transgene expression. Compared to cells from wild-type pigs, hPD-L1+ PBMC had a significantly reduced capacity to stimulate proliferation of human CD4+ T cells. Moreover, fibroblasts from hPD-L1 transgenic pigs were partially protected from cell-mediated lysis by human cytotoxic effector cells. CONCLUSIONS: These data indicate a low immunogenic, immune-protected status of cells from hPD-L1 transgenic pigs. The integration of the hPD-L1 concept into existing multi-transgenic pigs is promising to achieve long-term survival of porcine xenografts in non-human primate recipients.

Cyclic AMP Affects Oocyte Maturation and Embryo Development in Prepubertal and Adult Cattle.

High cAMP levels during in vitro maturation (IVM) have been related to improved blastocyst yields. Here, we employed the cAMP/cGMP modulators, forskolin, IBMX, and cilostamide, during IVM to unravel the role of high cAMP in early embryonic development produced from prepubertal and adult bovine oocytes. Oocytes were collected via transvaginal aspiration and randomly assigned to three experimental groups: TCM24 (24 h IVM/control), cAMP30 (2 h pre-IVM (forskolin-IBMX), 30 h IVM-cilostamide), and DMSO30 (Dimethyl Sulfoxide/vehicle control). After IVM, oocytes were fertilized in vitro and zygotes were cultured in vitro to blastocysts. Meiotic progression, cAMP levels, mRNA abundance of selected genes and DNA methylation were evaluated in oocytes. Blastocysts were used for gene expression or DNA methylation analyses. Blastocysts from the cAMP30 groups were transferred to recipients. The cAMP elevation delayed meiotic progression, but developmental rates were not increased. In immature oocytes, mRNA abundance of PRKACA was higher for cAMP30 protocol and no differences were found for PDE3A, SMAD2, ZAR1, PRDX1 and SLC2A8. EGR1 gene was up-regulated in prepubertal cAMP30 immature oocytes and down-regulated in blastocysts from all in vitro treatments. A similar gene expression profile was observed for DNMT3b, BCL2L1, PRDX1 and SLC2A8 in blastocysts. Satellite DNA methylation profiles were different between prepubertal and adult oocytes and blastocysts derived from the TCM24 and DMSO30 groups. Blastocysts obtained from prepubertal and adult oocytes in the cAMP30 treatment displayed normal methylation profiles and produced offspring. These data indicate that cAMP regulates IVM in prepubertal and adult oocytes in a similar manner, with impact on the establishment of epigenetic marks and acquisition of full developmental competency.

Risk of infection after iatrogenic perforation of the gut wall? Evaluation of preventive strategies in a randomized controlled animal trial.

BACKGROUND: Interventional endoscopies entail a risk of infection secondary to perforation of the luminal wall. Thereby, bacteria may be introduced into the sterile environment of the peritoneal cavity (PC). Limited data are available regarding the efficacy of prophylactic anti-infective treatments. The aim of the study was to examine the efficacy/safety of anti-infective means in the prevention of infection by interventional endoscopies in a randomized controlled animal trial. METHODS: Forty pigs were randomized to: 1: control; 2: oral lavage; 3: gastric lavage; 4: oral/gastric lavage; 5: i.m. antibiotics. Lavage was performed with Octenisept prior to the operation. After gastric wall perforation, peritoneoscopy was performed. Before the procedure, after closure and prior to autopsy, intraabdominal lavage for bacterial culture was taken using mini-laparoscopy. At autopsy, macroscopic appearance of the PC was scored. Lavage fluids were grown to identify/quantify bacterial load. Concentration of intraperitoneal bacteria at autopsy was defined as main outcome parameter. RESULTS: No major complications occurred in any of the procedures. Bacterial load of the PC at autopsy was significantly reduced with antibiotics compared to all other groups, whereas it did not differ between the lavage groups and control. Macroscopic scoring of the PC showed significant lower rate of intraabdominal abscesses in the antibiotic group compared to the lavage groups and control (p < 0.01). CONCLUSION: Only antibiotic prophylaxis is effective for the prevention of infection after iatrogenic perforation of the gastrointestinal wall. There was no difference between any form of lavage and the control group. Further studies in humans are required to prove these animal data.

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. In the absence of exogenous growth factors and/or small molecules, these inhibitors were able to maintain the expression of key pluripotency markers, including genes known to be specific for naive pluripotent state in mouse stem cells, for over 60 passages under feeder-free conditions. Surprisingly, the cells became dependent on HDAC inhibitors for the maintenance of proliferation. Moreover, despite showing successful integration into blastocysts upon injection, the cells were unable to undergo normal differentiation in vitro and in vivo in the form of teratomas. Our results suggest that HDAC inhibitors maintain pluripotency gene expression of porcine iPSC-like cells in long-term culture, but prevent lineage specification, requiring further optimization of culture conditions for porcine iPSC derivation.

Retracted article: In vitro derivation of mammalian germ cells from stem cells and their potential therapeutic application.

Pluripotent stem cells (PSCs) are a unique type of cells because they exhibit the characteristics of self-renewal and pluripotency. PSCs may be induced to differentiate into any cell type, even male and female germ cells, suggesting their potential as novel cell-based therapeutic treatment for infertility problems. Spermatogenesis is an intricate biological process that starts from self-renewal of spermatogonial stem cells (SSCs) and leads to differentiated haploid spermatozoa. Errors at any stage in spermatogenesis may result in male infertility. During the past decade, much progress has been made in the derivation of male germ cells from various types of progenitor stem cells. Currently, there are two main approaches for the derivation of functional germ cells from PSCs, either the induction of in vitro differentiation to produce haploid cell products, or combination of in vitro differentiation and in vivo transplantation. The production of mature and fertile spermatozoa from stem cells might provide an unlimited source of autologous gametes for treatment of male infertility. Here, we discuss the current state of the art regarding the differentiation potential of SSCs, embryonic stem cells, and induced pluripotent stem cells to produce functional male germ cells. We also discuss the possible use of livestock-derived PSCs as a novel option for animal reproduction and infertility treatment.

Heme Oxygenase-1 Gene Therapy Provides Cardioprotection Via Control of Post-Ischemic Inflammation: An Experimental Study in a Pre-Clinical Pig Model.

BACKGROUND: Heme oxygenase-1 (HO-1) is an inducible stress-responsive enzyme converting heme to bilirubin, carbon monoxide, and free iron, which exerts anti-inflammatory and antiapoptotic effects. Although efficient cardioprotection after HO-1 overexpression has been reported in rodents, its role in attenuating post-ischemic inflammation is unclear. OBJECTIVES: This study assessed the efficacy of recombinant adenoassociated virus (rAAV)-encoding human heme oxygenase-1 (hHO-1) in attenuating post-ischemic inflammation in a murine and a porcine ischemia/reperfusion model. METHODS: Murine ischemia was induced by 45 min of left anterior descending occlusion, followed by 24 h of reperfusion and functional as well as fluorescent-activated cell sorting analysis. Porcine hearts were subjected to 60 min of ischemia and 24h of reperfusion before hemodynamic and histologic analyses were performed. RESULTS: Human microvascular endothelial cells transfected with hHO-1 displayed an attenuated interleukin-6 and intercellular adhesion molecule 1 expression, resulting in reduced monocytic THP-1 cell recruitment in vitro. In murine left anterior descending occlusion and reperfusion, the post-ischemic influx of CD45(+) leukocytes, Ly-6G(+) neutrophils, and Ly-6C(high) monocytes was further exacerbated in HO-1-deficient hearts and reversed by rAAV.hHO-1 treatment. Conversely, in our porcine model of ischemia, the post-ischemic influx of myeloperoxidase-positive neutrophils and CD14(+) monocytes was reduced by 49% and 87% after rAAV.hHO-1 transduction, similar to hHO-1 transgenic pigs. Functionally, rAAV.hHO-1 and hHO-1 transgenic left ventricles displayed a smaller loss of ejection fraction than control animals. CONCLUSIONS: Whereas HO-1 deficiency exacerbates post-ischemic cardiac inflammation in mice, hHO-1 gene therapy attenuates inflammation after ischemia and reperfusion in murine and porcine hearts. Regional hHO-1 gene therapy provides cardioprotection in a pre-clinical porcine ischemia/reperfusion model.

Derivation and characterization of bovine induced pluripotent stem cells by transposon-mediated reprogramming.

Induced pluripotent stem cells (iPSCs) are a seminal breakthrough in stem cell research and are promising tools for advanced regenerative therapies in humans and reproductive biotechnology in farm animals. iPSCs are particularly valuable in species in which authentic embryonic stem cell (ESC) lines are yet not available. Here, we describe a nonviral method for the derivation of bovine iPSCs employing Sleeping Beauty (SB) and piggyBac (PB) transposon systems encoding different combinations of reprogramming factors, each separated by self-cleaving peptide sequences and driven by the chimeric CAGGS promoter. One bovine iPSC line (biPS-1) generated by a PB vector containing six reprogramming genes was analyzed in detail, including morphology, alkaline phosphatase expression, and typical hallmarks of pluripotency, such as expression of pluripotency markers and formation of mature teratomas in immunodeficient mice. Moreover, the biPS-1 line allowed a second round of SB transposon-mediated gene transfer. These results are promising for derivation of germ line-competent bovine iPSCs and will facilitate genetic modification of the bovine genome.

Effects of different oocyte retrieval and in vitro maturation systems on bovine embryo development and quality.

Cyclic adenosine monophosphate (cAMP) modulators have been used to avoid spontaneous oocyte maturation and concomitantly improve oocyte developmental competence. The current work evaluated the effects of the addition of cAMP modulators forskolin, 3-isobutyl-1-methylxanthine (IBMX) and cilostamide during in vitro maturation on the quality and yields of blastocysts. The following experimental groups were evaluated: (i) slicing or (ii) aspiration and maturation in tissue culture medium (TCM)199 for 24 h (TCM24slicing and TCM24aspiration, respectively), (iii) aspiration and maturation in the presence of cAMP modulators for 30 h (cAMP30aspiration) and in vivo-produced blastocysts. In vitro-matured oocytes were fertilized and presumptive zygotes were cultured in vitro to assess embryo development. Cleavage, blastocyst formation, blastocyst cell number, mRNA abundance of selected genes and global methylation profiles were evaluated. Blastocyst rate/zygotes for the TCM24aspiration protocol was improved (32.2 ± 2.1%) compared with TCM24slicing and cAMP30aspiration (23.4 ± 1.2% and 23.3 ± 2.0%, respectively, P 0.05), while those from the other groups were significantly elevated. It is concluded that retrieval, collection systems and addition of cAMP modulators can affect oocyte developmental competence, which is reflected not only in blastocyst rates but also in global DNA methylation and gene expression patterns.

MRTF-A controls vessel growth and maturation by increasing the expression of CCN1 and CCN2.

Gradual occlusion of coronary arteries may result in reversible loss of cardiomyocyte function (hibernating myocardium), which is amenable to therapeutic neovascularization. The role of myocardin-related transcription factors (MRTFs) co-activating serum response factor (SRF) in this process is largely unknown. Here we show that forced MRTF-A expression induces CCN1 and CCN2 to promote capillary proliferation and pericyte recruitment, respectively. We demonstrate that, upon G-actin binding, thymosin ß4 (Tß4), induces MRTF translocation to the nucleus, SRF-activation and CCN1/2 transcription. In a murine ischaemic hindlimb model, MRTF-A or Tß4 promotes neovascularization, whereas loss of MRTF-A/B or CCN1-function abrogates the Tß4 effect. We further show that, in ischaemic rabbit hindlimbs, MRTF-A as well as Tß4 induce functional neovascularization, and that this process is inhibited by angiopoietin-2, which antagonizes pericyte recruitment. Moreover, MRTF-A improves contractile function of chronic hibernating myocardium of pigs to a level comparable to that of transgenic pigs overexpressing Tß4 (Tß4tg). We conclude that MRTF-A promotes microvessel growth (via CCN1) and maturation (via CCN2), thereby enabling functional improvement of ischaemic muscle tissue.

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 Gsk3ß inhibitor) on the reprogramming of porcine fetal fibroblasts to induced pluripotent stem cells (piPSCs). Primary cultures treated with the two inhibitors (2i) showed a reduced number of alkaline phosphatase-positive colonies and a lower percentage of OCT4-expressing cells compared with the cultures grown with basic medium, which was supplemented with murine leukemia inhibitory factor (LIF). Moreover, the piPS-like cell lines established under 2i conditions expressed significantly lower levels of pluripotency markers, including OCT4, SOX2, REX1, UTF1, STELLA, TDH, and CHD1, compared with the controls. To test the short-term effects of the small molecule inhibitors, piPS-like cells that had been established in basic culture medium were cultured for five passages in medium supplemented with 2i or PD or CH individually. In accordance with the first experiment, expression levels of most pluripotency genes declined in cultures treated with inhibitors, although the response to each inhibitory molecule varied for the different genes. Results of this study concur with previous reports and cast doubts on the effectiveness of CH and PD in the reprogramming of porcine somatic cells to pluripotency.

Lack of antibody response in pigs immunized with the transmembrane envelope protein of porcine endogenous retroviruses.

Recently, we immunized different mammalian species (goats, mice, rats, rabbits, guinea pigs and hamsters) with the recombinant ectodomain of the transmembrane envelope (TM) protein p15E of porcine endogenous retrovirus (PERV). In all cases, neutralizing immune sera were induced, which recognized epitopes in the fusion peptide proximal region and the membrane proximal external region of p15E. In order to analyse whether pigs are also able to produce such antibodies, and whether such antibodies can be used to study the involvement of the TM protein in placental development (as was shown for endogenous retroviruses of other species), German landrace pigs were immunized with PERV p15E. No binding and neutralizing antibodies were produced as shown in three Western blot analyses and in a neutralization assay, indicating that pigs are tolerant to their endogenous retroviruses, at least for the ectodomain of the TM protein.

Promoter and lineage independent anti-silencing activity of the A2 ubiquitous chromatin opening element for optimized human pluripotent stem cell-based gene therapy.

Epigenetic silencing of retroviral transgene expression in pluripotent stem cells (PSC) and their differentiated progeny constitutes a major roadblock for PSC-based gene therapy. As ubiquitous chromatin opening elements (UCOEs) have been successfully employed to stabilize transgene expression in murine hematopoietic and pluripotent stem cells as well as their differentiated progeny, we here investigated UCOE activity in their human counterparts to establish a basis for future clinical application of the element. To this end, we demonstrate profound anti-silencing activity of the A2UCOE in several human iPS and ES cell lines including their progeny obtained upon directed cardiac or hematopoietic differentiation. We also provide evidence for A2UCOE activity in murine iPSC-derived hepatocyte-like cells, thus establishing efficacy of the element in cells of different germ layers. Finally, we investigated combinations of the A2UCOE with viral promoter/enhancer elements again demonstrating profound stabilization of transgene expression. In all these settings the effect of the A2UCOE was associated with strongly reduced promoter DNA-methylation. Thus, our data clearly support the concept of the A2UCOE as a generalized strategy to prevent epigenetic silencing in PSC and their differentiated progeny and strongly favors its application to stabilize transgene expression in PSC-based cell and gene therapy approaches.

A ubiquitous chromatin opening element prevents transgene silencing in pluripotent stem cells and their differentiated progeny.

Methylation-induced gene silencing represents a major obstacle to efficient transgene expression in pluripotent cells and thereof derived tissues. As ubiquitous chromatin opening elements (UCOE) have been shown to prevent transgene silencing in cell lines and primary hematopoietic cells, we hypothesized a similar activity in pluripotent cells. This concept was investigated in the context of cytidine deaminase (CDD) gene transfer, an approach to render hematopoietic cells resistant to the chemotherapeutic agent Ara-C. When murine induced pluripotent stem cells (iPSC)/embryonic stem cells (ESCs) were transduced with self-inactivating lentiviral vectors using housekeeping (truncated elongation factor 1α; EFS) or viral (spleen focus-forming virus; SFFV) promoters, incorporation of an heterogeneous nuclear ribonucleoproteins A2 B1/chromobox protein homolog 3 locus-derived UCOE (A2UCOE) significantly increased transgene expression and Ara-C resistance and effectively prevented silencing of the SFFV-promoter. The EFS promoter showed relatively stable transgene expression in naïve iPSCs, but rapid transgene silencing was observed upon hematopoietic differentiation. When combined with the A2UCOE, however, the EFS promoter yielded stable transgene expression in 73% ± 6% of CD41(+) hematopoietic progeny, markedly increased CDD expression levels, and significantly enhanced Ara-C resistance in clonogenic cells. Bisulfite sequencing revealed protection from differentiation-induced promoter CpG methylation to be associated with these effects. Similar transgene promoting activities of the A2UCOE were observed during murine neurogenic differentiation, in naïve human pluripotent cells, and during nondirected multilineage differentiation of these cells. Thus, our data provide strong evidence that UCOEs can efficiently prevent transgene silencing in iPS/ESCs and their differentiated progeny and thereby introduce a generalized concept to circumvent differentiation-induced transgene silencing during the generation of advanced iPSC/ESC-based gene and cell therapy products.

Derivation and characterization of sleeping beauty transposon-mediated porcine induced pluripotent stem cells.

The domestic pig is an important large animal model for preclinical testing of novel cell therapies. Recently, we produced pluripotency reporter pigs in which the Oct4 promoter drives expression of the enhanced green fluorescent protein (EGFP). Here, we reprogrammed Oct4-EGFP fibroblasts employing the nonviral Sleeping Beauty transposon system to deliver the reprogramming factors Oct4, Sox2, Klf4, and cMyc. Successful reprogramming to a pluripotent state was indicated by changes in cell morphology and reactivation of the Oct4-EGFP reporter. The transposon-reprogrammed induced pluripotent stem (iPS) cells showed long-term proliferation in vitro over >40 passages, expressed transcription factors typical of embryonic stem cells, including OCT4, NANOG, SOX2, REX1, ESRRB, DPPA5, and UTF1 and surface markers of pluripotency, including SSEA-1 and TRA-1-60. In vitro differentiation resulted in derivatives of the 3 germ layers. Upon injection of putative iPS cells under the skin of immunodeficient mice, we observed teratomas in 3 of 6 cases. These results form the basis for in-depth studies toward the derivation of porcine iPS cells, which hold great promise for preclinical testing of novel cell therapies in the pig model.