Oocytes, embryos and pluripotent stem cells from a biomedical perspective.

The veterinary and animal science professions are rapidly developing and their inherent and historical connection to agriculture is challenged by more biomedical and medical directions of research. While some consider this development as a risk of losing identity, it may also be seen as an opportunity for developing further and more sophisticated competences that may ultimately feed back to veterinary and animal science in a synergistic way. The present review describes how agriculture-related studies on bovine in vitro embryo production through studies of putative bovine and porcine embryonic stem cells led the way to more sophisticated studies of human induced pluripotent stem cells (iPSCs) using e.g. gene editing for modeling of neurodegeneration in man. However, instead of being a blind diversion from veterinary and animal science into medicine, these advanced studies of human iPSC-derived neurons build a set of competences that allowed us, in a more competent way, to focus on novel aspects of more veterinary and agricultural relevance in the form of porcine and canine iPSCs. These types of animal stem cells are of biomedical importance for modeling of iPSC-based therapy in man, but in particular the canine iPSCs are also important for understanding and modeling canine diseases, as e.g. canine cognitive dysfunction, for the benefit and therapy of dogs.

Identification of SSEA-1 expressing enhanced reprogramming (SEER) cells in porcine embryonic fibroblasts.

Previous research has shown that a subpopulation of cells within cultured human dermal fibroblasts, termed multilineage-differentiating stress enduring (Muse) cells, are preferentially reprogrammed into induced pluripotent stem cells. However, controversy exists over whether these cells are the only cells capable of being reprogrammed from a heterogeneous population of fibroblasts. Similarly, there is little research to suggest such cells may exist in embryonic tissues or other species. To address if such a cell population exists in pigs, we investigated porcine embryonic fibroblast populations (pEFs) and identified heterogeneous expression of several key cell surface markers. Strikingly, we discovered a small population of stage-specific embryonic antigen 1 positive cells (SSEA-1+) in Danish Landrace and Göttingen minipig pEFs, which were absent in the Yucatan pEFs. Furthermore, reprogramming of SSEA-1+ sorted pEFs led to higher reprogramming efficiency. Subsequent transcriptome profiling of the SSEA-1+ vs. the SSEA-1neg cell fraction revealed highly comparable gene signatures. However several genes that were found to be upregulated in the SSEA-1+ cells were similarly expressed in mesenchymal stem cells (MSCs). We therefore termed these cells SSEA-1 Expressing Enhanced Reprogramming (SEER) cells. Interestingly, SEER cells were more effective at differentiating into osteocytes and chondrocytes in vitro. We conclude that SEER cells are more amenable for reprogramming and that the expression of mesenchymal stem cell genes is advantageous in the reprogramming process. This data provides evidence supporting the elite theory and helps to delineate which cell types and specific genes are important for reprogramming in the pig.

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. The porcine stem cells used were: Induced pluripotent stem cells (iPSCs) created by lentiviral doxycycline-dependent reprogramming and cultered with a GSK3ß- and MEK-inhibitor (2i) and leukemia inhibitor factor (LIF) (2i LIF DOX-iPSCs), iPSCs created by a plasmid-based reprogramming and cultured with 2i and fibroblast growth factor (FGF) (2i FGF Pl-iPSCs) and embryonic germ cells (EGCs), which have earlier been characterized as being multipotent. The SCNT efficiencies of these stem cell lines were compared with that of the two fibroblast cell lines from which the iPSC lines were derived. The blastocyst rates for the 2i LIF DOX-iPSCs were 14.7%, for the 2i FGF Pl-iPSC 10.1%, and for the EGCs 34.5% compared with the fibroblast lines yielding 36.7% and 25.2%. The fibroblast- and EGC-derived embryos were used for embryo transfer and produced live offspring at similar low rates of efficiency (3.2 and 4.0%, respectively) and with several instances of malformations. In conclusion, potentially pluripotent porcine stem cells resulted in lower rates of embryonic development upon SCNT than multipotent stem cells and differentiated somatic cells.

Impaired APP activity and altered Tau splicing in embryonic stem cell-derived astrocytes obtained from an APPsw transgenic minipig.

Animal models of familial juvenile onset of Alzheimer's disease (AD) often fail to produce diverse pathological features of the disease by modification of single gene mutations that are responsible for the disease. They can hence be poor models for testing and development of novel drugs. Here, we analyze in vitro-produced stem cells and their derivatives from a large mammalian model of the disease created by overexpression of a single mutant human gene (APPsw). We produced hemizygous and homozygous radial glial-like cells following culture and differentiation of embryonic stem cells (ESCs) isolated from embryos obtained from mated hemizygous minipigs. These cells were confirmed to co-express varying neural markers, including NES, GFAP and BLBP, typical of type one radial glial cells (RGs) from the subgranular zone. These cells had altered expression of CCND1 and NOTCH1 and decreased expression of several ribosomal RNA genes. We found that these cells were able to differentiate into astrocytes upon directed differentiation. The astrocytes produced had decreased α- and ß-secretase activity, increased γ-secretase activity and altered splicing of tau. This indicates novel aspects of early onset mechanisms related to cell renewal and function in familial AD astrocytes. These outcomes also highlight that radial glia could be a potentially useful population of cells for drug discovery, and that altered APP expression and altered tau phosphorylation can be detected in an in vitro model of the disease. Finally, it might be possible to use large mammal models to model familial AD by insertion of only a single mutation.

Isolation and culture of porcine neural progenitor cells from embryos and pluripotent stem cells.

The isolation and culture of neural progenitor cells (NPCs) from pluripotent stem cells has facilitated in vitro mechanistic studies of diseases related to the nervous system, as well as discovery of new medicine. In addition, NPCs are envisioned to play a crucial role in future cell replacement therapy. The pig has become recognized as an important large animal model and establishment of in vitro-derived porcine NPCs would allow for preclinical safety testing by transplantation in a porcine biomedical model. In this chapter, a detailed method for isolation and in vitro culture of porcine NPCs from porcine embryos or induced pluripotent stem cells is presented. The neural induction is performed in coculture and the isolation of rosette structures is carried out manually to ensure a homogenous population of NPCs. Using this method, multipotent NPCs can be obtained in approximately 1 month. The cells have the potential of long-term culture and the ability to differentiate into neural and glial cells.

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.

Temporal repression of endogenous pluripotency genes during reprogramming of porcine induced pluripotent stem cells.

Porcine induced pluripotent stem cells (piPSCs) have the capacity to differentiate in vitro and in vivo and form chimeras. However, the lack of transgene silencing of exogenous DNA integrated into the genome and the inability of cells to proliferate in the absence of transgene expression are underlying reported problems, suggesting that reprogramming is not complete. The aim of the present study was to evaluate reprogramming events using a partially reprogrammed piPSC-like line expressing hOCT4, hNANOG, and hcMYC under tetracycline-regulated control to investigate the effects of these particular transgenes on the expression of the porcine endogenous pluripotency machinery. Endogenous and exogenous gene expression of OCT4, NANOG, SOX2, KLF4, and cMYC was determined at passages 5, 10, 15, and 20, both in cells cultured at 1 µg/mL doxycycline or 4 µg/mL doxycycline. Our results revealed that endogenous genes are repressed by their transgene counterparts in culture and that lack of expression of the transgenes, SOX2 and KLF4 allows for expression of endogenous SOX2 and KLF4. Furthermore, we report that alternate endogenous transcripts for pNANOG, pSOX2, and pKLF4 can also be detected in the pig. Despite the ability for some endogenous genes to be expressed in these lines, the piPSC-like cells still cannot be maintained without doxycycline, indicating that the culture system of piPSCs may not be optimal or that the reprogramming factor combination used may not currently be optimal for maintaining pluripotency in the pig. This may help to explain the difficulties in producing stable piPSCs and bona fide embryonic stem cell lines in this species.

Identification of molecules derived from human fibroblast feeder cells that support the proliferation of human embryonic stem cells.

The majority of human embryonic stem cell lines depend on a feeder cell layer for continuous growth in vitro, so that they can remain in an undifferentiated state. Limited knowledge is available concerning the molecular mechanisms that underlie the capacity of feeder cells to support both the proliferation and pluripotency of these cells. Importantly, feeder cells generally lose their capacity to support human embryonic stem cell proliferation in vitro following long-term culture. In this study, we performed large-scale gene expression profiles of human foreskin fibroblasts during early, intermediate and late passages using a custom DNA microarray platform (NeuroStem 2.0 Chip). The microarray data was validated using RT-PCR and virtual SAGE analysis. Our comparative gene expression study identified a limited number of molecular targets potentially involved in the ability of human neonatal foreskin fibroblasts to serve as feeder cells for human embryonic stem cell cultures. Among these, the C-KIT, leptin and pigment epithelium-derived factor (PEDF) genes were the most interesting candidates.

Ultrastructural and molecular distinctions between the porcine inner cell mass and epiblast reveal unique pluripotent cell states.

Characterization of the pluripotent cell populations within the porcine embryo is essential for understanding pluripotency and self-renewal regulation in the inner cell mass (ICM) and epiblast. In this study, we perform detailed ultrastructural and molecular characterization of the developing pluripotent cell population as it develops from the ICM to the late epiblast. The ultrastructural observations revealed that the outer cells of the ICM have a high nuclear:cytoplasmic ratio but are transcriptionally inactive and contain mitochondria with few cristae. In contrast, the epiblast cells have a reduced nuclear:cytoplasmic ratio, are more transcriptionally active, and contain abundant cellular organelles. This study also revealed cavitation and potential unfolding of the epiblast. As the ICM forms the epiblast, SSEA1 is lost and VIMENTIN is lost and re-expressed. The D6 blastocyst expressed high levels of STELLA, TERF1, and GDF3, and the epiblast expressed epithelial markers, MUC1 and E-CADHERIN, and the pluripotency markers, DNMT3B and CRIPTO.

Porcine pluripotency cell signaling develops from the inner cell mass to the epiblast during early development.

The signaling mechanisms regulating pluripotency in porcine embryonic stem cells and embryos are unknown. In this study, we characterize cell signaling in the in-vivo porcine inner cell mass and later-stage epiblast. We evaluate expression of OCT4, NANOG, SOX2, genes within the JAK/STAT pathway (LIF, LIFR, GP130), FGF pathway (bFGF, FGFR1, FGFR2), BMP pathway (BMP4), and downstream-activated genes (STAT3, c-Myc, c-Fos, and SMAD4). We discovered two different expression profiles exist in the developing porcine embryo. The D6 porcine blastocyst (inner cell mass stage) is devoid in the expression of most genes analyzed, with the exception of OCT4. In contrast, the D11 epiblast expressed 10 of the 12 genes investigated. Immunocytochemistry confirmed LIFR and bFGF was not expressed in the epiblast, but within the trophectoderm. These findings reveal cell signaling associated with maintaining pluripotency in human embryonic stem cells is detectable in the porcine epiblast, but not in the inner cell mass.

Restorative cell therapy for Parkinson's disease: a quest for the perfect cell.

The development of a cell therapy for the neurodegenerative disorder Parkinson's disease is a realistic ambition. It is pursued by researchers and companies alike, and spans different donor tissue types of embryonic, fetal and adult origins. In this review, we briefly outline the past and current status of research and clinical trials with cell transplantation in Parkinson's disease. We discuss studies on donor tissue derived from embryonic ventral mesencephalon and assess the current research on various forms of stem cells of both embryonic and adult origins in the quest to develop a cell-based therapy for this debilitating movement disorder.

Using therapeutic cloning to fight human disease: a conundrum or reality?

The development and transplantation of autologous cells derived from nuclear transfer embryonic stem cell (NT-ESC) lines to treat patients suffering from disease has been termed therapeutic cloning. Human NT is still a developing field, with further research required to improve somatic cell NT and human embryonic stem cell differentiation to deliver safe and effective cell replacement therapies. Furthermore, the implications of transferring mitochondrial heteroplasmic cells, which may harbor aberrant epigenetic gene expression profiles, are of concern. The production of human NT-ESC lines also remains plagued by ethical dilemmas, societal concerns, and controversies. Recently, a number of alternate therapeutic strategies have been proposed to circumvent the moral implications surrounding human nuclear transfer. It will be critical to overcome these biological, legislative, and moral restraints to maximize the potential of this therapeutic strategy and to alleviate human disease.

Production of a cloned calf using zona-free serial nuclear transfer.

The efficiency of generating cloned animals following somatic cell nuclear transfer appears to have reached a plateau, despite ongoing research to improve developmental outcomes. A major limitation appears in the restricted nature of the adult/donor cell to de-differentiate to form a totipotent nucleus. Serial nuclear transfer, a modified cloning technique, has increased the developmental competence of amphibian, murine and porcine cloned embryos. This procedure involves a second nuclear transfer step; pronuclear-like cloned nuclei are transferred into pronuclear stage zygotic cytoplasts. The present study reports on the development of a serial nuclear transfer technique in the bovine, based on a zona-free method (hand-made cloning), resulting in the birth of a cloned calf. Comparisons were made between embryos produced by hand-made cloning and serial nuclear transfer. There were no differences between in vitro development or differential cell counts in the blastocysts produced. Transfer of 16 serial hand-made cloned blastocysts resulted in the production of one healthy calf (6%), whereas hand-made cloning resulted in the birth of 1 calf from 23 transferred blastocysts (4%). One serial nuclear transfer pre-term fetus had renal and hepatic abnormalities (previously observed in clones from this cell line). Although it may not be as beneficial in the bovine as in other species, normal placentation (size, placentomes and umbilicus) was encouraging. Refinement of this technique may help to identify species-specific differences in zygotic competence that affect reprogramming of donor cell nuclei and that may improve efficiency.

The status of human nuclear transfer.

Human therapeutic cloning is a recently emerged application of somatic cell nuclear transfer (SCNT), which is currently being performed to produce patient-specific stem cell lines for future stem cell therapies. The advantages in producing human nuclear transfer (NT) embryos to derive NT stem cell lines are that these can be tailor-made (i.e., are autologous in nature) for the patient and may overcome the need to administer life-long immunosuppression following stem cell transplantation. Although the rationale for using NT embryos is not for reproductive purposes, human NT remains clouded in ethical, moral, and religious controversies. The recent retraction of high-impact factor publications in the field of human NT from a research group in South Korea has placed stem cell research in a delicate situation. These heavily publicized issues may hinder the progress of this research and may threaten to bring current research to a complete halt. This review outlines the recent status of human NT, its continuing progress and the difficulties the field faces. Of most concern are the ethical issues, which surround obtaining human oocytes for research. Recent evidence suggests that failed-to-fertilize oocytes are poor sources for human SCNT, but obtaining fresh, viable oocytes may be even more problematic. The current status of human SCNT is outlined in this review with particular reference made to, lessons learnt from animal research, the oocyte dilemma and optimization of human NT.

Nuclear lamin antigen and messenger RNA expression in bovine in vitro produced and nuclear transfer embryos.

The nuclear lamina is a complex meshwork of nuclear lamin filaments that lies on the interface of the nuclear envelope and chromatin and is important for cell maintenance, nucleoskeleton support, chromatin remodeling, and protein recruitment to the inner nucleolus. Protein and mRNA patterns for the major nuclear lamins were investigated in bovine in vitro fertilized (IVF) and nuclear transfer embryos. Expression of lamins A/C and B were examined in IVF bovine germinal vesicle (GV) oocytes, metaphase II oocytes, zygotes, 2-cell, 8-cell, 16-32-cell embryos, morulae, and blastocysts (n = 10). Lamin A/C was detected in 9/10 immature oocytes, 10/10 zygotes, 8/10 2-cell embryos, 4/10 morulae, 10/10 blastocysts but absent during the maternal embryonic transition. Lamin B was ubiquitously expressed during IVF preimplantation development but was only detected in 4/10 GV oocytes. Messenger RNA expression confirms that the major lamins, A/C and B1 are expressed throughout preimplantation development and transcribed by the embryo proper. Lamin A/C and B expression were observed (15 min, 30 min, 60 min, 120 min) following somatic cell nuclear transfer using adult fibroblasts and at the 2-cell, 8-cell, 16-32-cell, morula and blastocyst stage (n = 5). Altered expression levels and localization of nuclear lamins A/C and B was determined in nuclear transfer embryos during the first 2 hr post fusion, coincidental with only partial nuclear envelope breakdown as well as during the initial cleavage divisions, but was restored by the morula stage. This mechanical and molecular disruption of the nuclear lamina provides key evidence for incomplete nuclear remodeling and reprogramming following somatic cell nuclear transfer.

Expression profiling of genes crucial for placental and preimplantation development in bovine in vivo, in vitro, and nuclear transfer blastocysts.

Placental abnormalities and failed implantation are characterized phenotypes that occur in many species as a result of somatic cell cloning. This study examines a number of genes, critical for early placental development and reports aberrant expression patterns in a number of cloned bovine blastocysts, thus implicating a role of these genes in failed implantation. Messenger RNA (mRNA) expression of eight genes critical for early placental and preimplantation development including Acrogranin, Cdx2, Eomes, ErbB3, ERR2, Hand1, MRJ, and Rex1 were analyzed in single, in vivo, in vitro, and cloned bovine blastocysts (produced by hand-made cloning (HMC) and serial hand-made cloning (SHMC)) following complementary DNA (cDNA) amplification with a SMART cDNA synthesis kit. Aberrant expression of Acrogranin, Cdx2, and ERR2 was detected in a number of blastocysts produced by SHMC. Other genes, Eomes and Hand1, were not detectable in, in vivo bovine blastocysts, suggesting a differential expression pattern between bovine and murine embryos. A number of control marker genes including Oct4, IFN-tau, and PolyA were expressed in all single blastocysts analyzed. This is the first study to report that failure of implantation may be due to aberrant expression of genes in the preimplantation cloned embryo, which are crucial for the early regulation and differentiation of the placenta.

Effect of exogenous DMNPE-caged ATP on in vitro-matured bovine oocytes prior to parthenogenetic activation, fertilisation and nuclear transfer.

Adenosine triphosphate (ATP) plays an important role during fertilisation of the mammalian oocyte through its ability to alter the frequency and duration of calcium oscillations. It has also been shown that higher ATP levels correlate with increased developmental competence in bovine and human oocytes. During somatic cell nuclear transfer (NT), the incoming nucleus is remodelled extensively, undoubtedly using a variety of ATP-dependent enzymes. The aim of the present study was to determine whether additional exogenous ATP influences activation of parthenogenetic (PA), in vitro-fertilised (IVF) or cloned (NT) in vitro-matured bovine oocytes. Blastocyst development and cell numbers in PA embryos were found to increase in a dose-dependent manner following the photorelease of 0, 50, 100, 500 and 1000 microm DMNPE-caged ATP (adenosine 5'-triphosphate, P3-(1-(4,5-dimethoxy-2-nitrophenyl)ethyl) ester, disodium salt). No cleavage was found following release of 2 and 5 mm DMNPE-caged ATP or with DMNPE-caged ATP (not photoreleased). There were also no differences in blastocyst rates or cell numbers between the control group and groups treated with caged, but not photoreleased, ATP. The addition of exogenous ATP before IVF or to NT couplets did not result in a significant increase in blastocyst development or cell number. Embryo transfer is necessary to determine whether exogenous ATP can positively affect reprogramming, resulting in higher cloned pregnancy rates or live-term births.