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.

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.

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.

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.

In vivo oocyte developmental competence is reduced in lean but not in obese superovulated dairy cows after intraovarian administration of IGF1.

The present study investigated the role of IGF1 in lactating lean and non-lactating obese dairy cows by injecting 1 µg IGF1 into the ovaries prior to superovulation. This amount of IGF1 has been linked with pregnancy loss in women with the polycystic ovary syndrome (PCOS) and was associated with impaired bovine oocyte competence in vitro. Transcript abundance and protein expression of selected genes involved in apoptosis, glucose metabolism, and the IGF system were analyzed. Plasma concentrations of IGF1 and leptin, and IGF1 in uterine luminal fluid (ULF), were also measured. IGF1 treatment decreased embryo viability in lean cows to the levels observed in obese cows. Obese cows were not affected by IGF1 treatment and showed elevated levels of IGF1 (in both plasma and ULF) and leptin. Blastocysts from lean cows treated with IGF1 showed a higher abundance of SLC2A1 and IGFBP3 transcripts. IGF1 treatment reduced protein expression of tumor protein 53 in blastocysts of lean cows, whereas the opposite was observed in obese cows. IGF1 in plasma and ULF was correlated only in the control groups. Blastocyst transcript abundance of IGF1 receptor and IGFBP3 correlated positively with IGF1 concentrations in both plasma and ULF in lean cows. The detrimental microenvironment created by IGF1 injection in lean cows and the lack of effect in obese cows resemble to a certain extent the situation observed in PCOS patients, where IGF1 bioavailability is increased in normal-weight women but reduced in obese women, suggesting that this bovine model could be useful for studying IGF1 involvement in PCOS.

Oct4-enhanced green fluorescent protein transgenic pigs: a new large animal model for reprogramming studies.

The domesticated pig has emerged as an important tool for development of surgical techniques, advancement of xenotransplantation, creation of important disease models, and preclinical testing of novel cell therapies. However, germ line-competent pluripotent porcine stem cells have not yet been derived. This has been a major obstacle to genetic modification of pigs. The transcription factor Oct4 is essential for the maintenance of pluripotency and for reprogramming somatic cells to a pluripotent state. Here, we report the production of transgenic pigs carrying an 18 kb genomic sequence of the murine Oct4 gene fused to the enhanced green fluorescent protein (EGFP) cDNA (OG2 construct) to allow identification of pluripotent cells by monitoring Oct4 expression by EGFP fluorescence. Eleven viable transgenic piglets were produced by somatic cell nuclear transfer. Expression of the EGFP reporter construct was confined to germ line cells, the inner cell mass and trophectoderm of blastocysts, and testicular germ cells. Reprogramming of fibroblasts from these animals by fusion with pluripotent murine embryonic stem cells or viral transduction with human OCT4, SOX2, KLF4, and c-MYC cDNAs resulted in Oct4-EGFP reactivation. The OG2 pigs have thus proved useful for monitoring reprogramming and the induction and maintenance of pluripotency in porcine cells. In conclusion, the OG2 transgenic pigs are a new large animal model for studying the derivation and maintenance of pluripotent cells, and will be valuable for the development of cell therapy.

DNA methylation patterns reflect epigenetic reprogramming in bovine embryos.

To understand the epigenetic alterations associated with assisted reproduction technology (ART) and the reprogramming of gene expression that follows somatic cell nuclear transfer (SCNT), we screened a panel of 41 amplicons representing 25 developmentally important genes on 15 different chromosomes (a total of 1079 CpG sites). Methylation analysis was performed on DNA from pools of 80 blastocysts representing three classes of embryos. This revealed a subset of amplicons that distinguish between embryos developing in vivo, produced in vitro, or reconstructed by SCNT. Following SCNT, we observed massive epigenetic reprogramming evidenced by reduced levels of methylation in the resultant embryos. Analysis of data from the 28 most informative amplicons (hotspot loci), representing more than 523 individual CpG sites, we discovered subsets of amplicons with methylation patterns that were unique to each class of embryo and may indicate metastable epialleles. Analysis of eight genes with respect to mRNA expression did not reveal a direct correlation with DNA methylation levels. In conclusion, this approach revealed a subset of amplicons that can be used to evaluate blastocyst quality and reprogramming following SCNT, and can also be employed for the localization of the epigenetic control regions within individual genes and for more general studies of stem cell differentiation.

Transgenic expression of the human A20 gene in cloned pigs provides protection against apoptotic and inflammatory stimuli.

BACKGROUND: Porcine organs with transgenic expression of anti-apoptotic and anti-inflammatory genes like the human A20 gene (hA20), a tumor necrosis factor-alpha (TNF-alpha)-inducible gene, may control the acute vascular rejection (AVR) of porcine xenografts. The human A20 molecule possesses protective features against inflammatory and apoptotic stimuli in various cell types including endothelial cells, rendering it a promising candidate for transgenic pig production in the context of xenotransplantation. Here, we produced pigs transgenic for hA20 and investigated whether hA20-transgenic porcine aortic endothelial cells (PAECs) were resistant against the induction of apoptosis in vitro and to what extent hA20-transgenic porcine hearts were protected against ischemia/reperfusion (I/R) injury. METHODS: Porcine fetal fibroblasts (PFFs) were transfected with the vector pCAGGSEhA20-IRESNEO containing a chicken beta-actin/rabbit beta-globin (CAGGS)-promoter element, known to provide ubiquitous gene expression in both mice and pigs. Transfected PFFs were then used in somatic cell nuclear transfer (SCNT). Three hA20-transgenic pigs were killed for PAEC isolation and organ mRNA and protein expression analysis by reverse transcriptase-polymerase chain reaction (RT-PCR), Northern and Western Blotting. PAECs were tested for susceptibility to apoptosis after TNF-alpha challenging and triggering of the CD95(Fas)/CD95Ligand pathway. Five transgenic and three wild type animals were subjected to an I/R experiment followed by measurement of infarct size, myeloperoxidase (MPO) activity and subendocardial segmental shortening (SES) to assess protective effects of hA20 in the porcine myocardium. RESULTS: The hA20-transgenic pigs developed normally and expression of hA20 was found in skeletal muscle, heart and PAECs. Cultured human A20-transgenic PAECs showed significantly reduced apoptosis when compared to their wild type counterparts and were less susceptible to the induction of cell death by CD95(Fas)L. Only partial protection of hA20-transgenic pig hearts was observed after I/R. While infarct size did not differ between the two groups after ischemic assault, hA20-transgenic porcine hearts showed significantly lower MPO activity and better hemodynamic performance (determined as SES) than their wild type counterparts. CONCLUSIONS: The hA20 gene was for the first time functionally expressed in transgenic pigs. Although the CAGGS is a ubiquitous promoter element, expression was restricted to heart, skeletal muscle and PAECs of transgenic animals. Cultivated hA20-transgenic PAECs were protected against TNF-alpha-mediated apoptosis, and partially protected against CD95(Fas)L-mediated cell death; cardiomyocytes were partially protected in I/R. These findings reveal hA20 as a promising molecule for controlling AVR in multi-transgenic pigs for xenotransplantation studies.

Effects of insulin-like growth factor-1 on cellular and molecular characteristics of bovine blastocysts produced in vitro.

Addition of insulin-like growth factor-1 (IGF-1) to culture medium increases the proportion of bovine embryos that develop to the blastocyst stage and increases embryo survival following transfer to heat-stressed, lactating dairy cows. The objective of the present study was to determine molecular and cellular correlates of these actions of IGF-1. Embryos were produced in vitro and cultured for 7 days with or without 100 ng/ml IGF-1. On d 7 after insemination, grade 1 expanded blastocysts were harvested and used to determine total cell number, percent apoptosis, cell allocation to the inner cell mass and trophectoderm, and the relative abundance of several developmentally important gene transcripts. There was no significant effect of IGF-1 treatment on blastocyst cell number, the proportion of blastomeres that were apoptotic, or the number of cells in the inner cell mass and trophectoderm. However, differences in the relative abundance of several mRNA transcripts were observed between control and IGF-1 treated embryos. Addition of IGF-1 increased (P < 0.02) amounts of mRNA for IGF binding protein-3 and desmocollin II and tended (P < 0.08) to increase amounts of mRNA for Na/K ATPase and Bax. Moreover, IGF-1 treatment decreased (P < 0.05) steady-state amounts of transcripts for heat shock protein 70 and tended (P < 0.08) to reduce amounts of IGF-1 receptor mRNA. In conclusion, increased survival of embryos treated with IGF-1 does not appear due to effects on cell number, percent apoptosis, or cell allocation. Addition of IGF-1 to culture can, however, alter expression of several transcripts which may be important for embryo development and survival following transfer.

Effect of CD34 cell dose on hematopoietic reconstitution and outcome in 508 patients with multiple myeloma undergoing autologous peripheral blood stem cell transplantation.

BACKGROUND: We analyzed the hematopoietic reconstitution and outcome of 508 patients with multiple myeloma (MM) with respect to the number of CD34+ cells reinfused at our center. PATIENTS AND METHODS: Each cohort of 390 patients (unselected CD34+ cell transplant) and 118 patients (CD34+ selected transplant) was divided into four subgroups. Among the 390 transplantations, 86 patients received a high dose (HD-) of > or =6.50 x 10(6) unselected CD34+ cells/kg, 116 patients a low dose (LD-) of <3.00 x 10(6) CD34+ cells/kg. Among the patients treated with CD34+ selected PBSC, 34 received > or =6.50 x 10(6) CD34+ cells/kg (HD+) and 16 <3.00 x 10(6) CD34+ cells/kg (LD+). RESULTS: HD- patients experienced a reduced median time to leukocyte (13 d vs. 14 d) (P < 0.001) and platelet reconstitution >20 x 10(9)/L (10 d vs. 12 d) (P < 0.001). Similarly, HD+ showed a reduced median time to leukocyte (12 d vs. 15 d) (P < 0.001) and platelet recovery >20 x 10(9)/L (10 d vs. 11 d) (P = 0.058). CD34+ cell-dose was significant for long-term platelet recovery at day 360 (unselected transplant P = 0.015, selected transplant P = 0.023). Number of transplanted CD34+ cells had no significant impact on transplant related mortality, overall survival or CR/PR rates within 100 d. In terms of supportive care the differences of high-/low-dose grafts were minimal. CONCLUSIONS: These results confirm that high doses of CD34+ PBSC shorten hematopoietic reconstitution and reduce hospitalization. Nevertheless secure engraftment results from transplantation of 2.00-3.00 x 10(6) CD34+ cells/kg. As 60% of our pretreated patients are able to collect > or =5.00 x 10(6) CD34+ cells/kg within a single leukapheresis, division into two or more freezing bags allows safe tandem transplantation in the majority of MM patients.

Kinetics of peripheral blood stem cell mobilization following G-CSF-supported chemotherapy.

It would be a clinical and economical advantage if the optimal time point of peripheral blood stem cell (PBSC) mobilization following G-CSF-supported chemotherapy (CT) was known in advance. Therefore, we retrospectively analyzed mobilization parameters in 113 adult tumor patients treated in our institution within 1 year. The start of apheresis was guided by CD34(+) cell measurements in the PB and occurred on or after day 11 after start of mobilization CT in 97% of patients. The median peak (p)CD34(+) cell count in PB uniformly occurred on day 14-15 (range: 6-32 days) after the start of CT, irrespective of the diagnosis (multiple myeloma n = 76, other histology n = 37), the type, but not the amount, of premobilization CT or radiotherapy (RT), the mobilization regimen, or the G-CSF dosage administered. Among more heavily pretreated patients (>six cycles of prior CT or RT), a higher proportion mobilized late (pCD34(+) cell count later than day 20 in 12% and 13%, respectively, versus 2%-5% in the other groups). Therefore, we propose to start measuring CD34(+) cells in the PB on day 11 after the start of mobilization therapy. The wide range of optimal mobilization time points argues for an individualized rather than a preset start of apheresis.

Laparoscopical intrauterine insemination with different doses of fresh, conserved, and frozen semen for the production of ovine zygotes.

The objective of the present study was to increase the efficiency in the production of ovine zygotes suitable for microinjection via laparoscopical intrauterine insemination. In the first part of the study, 71 ewes of three different breeds were inseminated with one of two different insemination doses (50 x 10(6) or 300 x 10(6) sperm per inseminate) and semen was either freshly diluted, liquid conserved, or frozen/thawed, or females were mated by a fertile ram (controls). In the second part, a total of 46 ewes was inseminated with 300 x 10(6) freshly diluted sperm to verify the findings from part 1 and to unravel effects of breed and age of donor ewe. The oviducts were flushed 24-26 h after insemination and the success of insemination was assessed by microscopical examination. Recovery rates were 78.0+/-26.4 and 72.1+/-24.6% in parts 1 and 2 of the study, respectively. Of these oocytes 62.3 and 62.8% (parts 1 and 2, respectively) were fertilized. In part 1, the highest proportion (64.7%) of pronuclear stages was observed in the group inseminated with 300 x 10(6) freshly diluted semen and was significantly higher compared to the groups inseminated with 50 x 10(6) freshly diluted semen (25.5%, P<0.001), 300 x 10(6) liquid conserved semen (49.0%, P<0.001), or 50 x 10(6) frozen/thawed semen (39.6%, P<0.05). In the control group, the proportion of pronuclear stages amounted to 60.2%. Irrespective of the type of sperm conservation, the overall fertilization rate (zygotes plus 2-cell stages) was higher (P<0.05) following insemination with 300 x 10(6) sperm (68.2%) compared to 50 x 10(6) sperm (56.8%). In part 2, the proportion of pronuclear stages reached 54.2% with an overall fertilization rate of 62.9%. These results were affected by breed and age of the donor as crossbred and younger (<3 years) animals yielded the highest proportion of pronuclear stages. The present study shows that freshly diluted semen at a dosage of 300 x 10(6) sperm yields the highest fertilization rates, the greatest proportion of pronuclear stages and the lowest proportion of mature unfertilized oocytes. Further increases in yields of pronuclear stages can possibly be achieved by selection of sheep from the best suited breed and younger than 3 years of age.