Ivan Vassiliev

Establishment of Pluripotent Cell Lines from Vertebrate Species – Present Status and Future Prospects

Pluripotent embryonic stem (ES) cells are undifferentiated cell lines derived from early embryos and are capable of unlimited undifferentiated proliferation in vitro. They retain the ability to differentiate into all cell types including germ cells in chimeric animals in vivo, and can be induced to form derivatives of all three germ layers in vitro. Mouse ES cells represent one of the most important tools in genetic research. Major applications include the targeted mutation of specific genes by homologous recombination and the discovery of new genes by gene trap strategies. These applications would be of high interest for other model organisms and also for livestock species. However, in spite of tremendous research activities, no proven ES cells colonizing the germ line have been established for vertebrate species other than mouse and chicken thus far. This review summarizes the current status of deriving pluripotent embryonic stem cell lines from vertebrates and recent developments in nuclear transfer technology, which may provide an alternative tool for genetic modification of livestock animals.

In Vitro and In Vivo Characterization of Putative Porcine Embryonic Stem Cells

We have developed a new method for the isolation of porcine embryonic stem cells (ESCs) from in vivo-derived and in vitro-produced embryos. Here we describe the isolation and characterization of several ESC lines established using this method. Cells from these lines were passaged up to 14 times, during which they were repeatedly cryopreserved. During this time, ESCs maintained their morphology and continued to express Oct 4, Nanog, and SSEA1. These cells formed embryoid bodies in suspension culture, and could be directed to differentiate into various lineages representative of all three germ layers in vitro. When injected into blastocysts these cells localized in the inner cell mass of blastocysts. To examine their pluripotency further, cells were injected into host blastocysts and transferred to recipient animals. Of the six transfers undertaken, one recipient became pregnant and gave birth to a litter of one male and three female piglets. Microsatellite analysis of DNA extracted from the tail tissue of these piglets indicated that two female piglets were chimaeric.

Isolation and in vitro characterization of putative porcine embryonic stem cells from cloned embryos treated with trichostatin A.

We report here the establishment and characterization of putative porcine embryonic stem cell (ESC) lines derived from somatic cell nuclear transfer embryos (NT-ESCs). These cells had a similar morphology to that described previously by us for ESCs derived from in vitro produced embryos, namely, a polygonal shape, a relatively small (10-15 μm) diameter, a small cytoplasmic/nuclear ratio, a single nucleus with multiple nucleoli and multiple lipid inclusions in the cytoplasm. NT-ESCs could be passaged at least 15 times and vitrified repeatedly without changes in their morphology, karyotype, or Oct-4 and Nanog expression. These cells formed embryoid bodies and could be directed to differentiate in vitro to cell types representative of all three germ layers. Following their injection into blastocysts, these cells preferentially localized in the inner cell mass. In conclusion, we have isolated putative porcine ESCs from cloned embryos that have the potential to be used for a variety of applications including as a model for human therapeutic cloning.

Insulin increases epiblast cell number of in vitro cultured mouse embryos via the PI3K/GSK3/p53 pathway.

High-quality embryos give rise to embryonic stem cells (ESCs) at greater efficiencies than poor-quality embryos. However, most embryos available for human ESC derivation are of a reduced quality as a result of culture in relatively simple media up to 10 years earlier, before cryopreservation, or before compaction. In the present study, we used a mouse model to determine whether a culture with insulin from the 8-cell stage could increase the number of ESC progenitor epiblast cells in blastocysts, as well as endeavor to determine the molecular mechanism of the insulins effect. Culture in media containing 1.7 ρM insulin increased epiblast cell number (determined by Oct4 and Nanog co-expression), and proportion in day 6 blastocysts. The inhibition of phosphoinositide 3 kinase (PI3K) (via LY294002), an early second messenger of the insulin receptor, blocked this effect. The inhibition of glycogen synthase kinase 3 (GSK3) or p53, 2 s messengers inactivated by insulin signaling (via CT99021 or pifithrin-α, respectively), increased epiblast cell numbers. When active, GSK3 and p53 block the transcription of Nanog, which is important for maintaining pluripotency. A simultaneous inhibition of GSK3 and p53 had no synergistic effects on epiblast cell number. The induced activation of GSK3 and p53, via the inhibition of proteins responsible for their inactivation (PKA via H-89 and SIRT-1 via nicotinamide, respectively), blocked the insulins effect on the epiblast.From our findings, we conclude that insulin increases epiblast cell number via the activation of PI3K, which ultimately inactivates GSK3 and p53. Furthermore, we suggest that the inclusion of insulin in culture media could be used as a strategy for increasing the efficiency with which the ESC lines can be derived from cultured embryos.

On the need for porcine embryonic stem cells to produce Gal KO pigs expressing multiple transgenes to advance xenotransplantation research

In reviewing the transgenesis literature in 2001, we concluded that while gene targeting and nuclear transfer of somatic cells bypassed the need for porcine embryonic stem cells (pESCs) to generate a1,3 galactosyltransferase knockout (Gal KO) pigs, the limited lifespan of these cells in culture prevented us from performing multiple rounds of homologous recombination to produce Gal KO pigs expressing multiple transgenes inserted as knockins in the space of one generation [1]. In particular, we concluded that a cell type ideally pESCs would still be required if such animals were to be produced without having to resort to generations of breeding and that this was a major technical hurdle because it had been suggested that as many as 10 genes may need to be examined before clinical trials of xenotransplantation could be contemplated [reviewed in 2]. Almost 10 years on from our initial review, the question needs to be asked: just how far are we from achieving this goal?

Isolation and Culture of Porcine Embryonic Stem Cells

Despite their agricultural and biomedical importance, embryonic stem cells (ESCs) are yet to be isolated for the pig or the domestic ungulates in general. This suggests that methods which have been used successfully in mice may not be applicable to these. In this chapter we describe a new method for the isolation of porcine ESCs. This method differs from those described previously in that it produces homogeneous outgrowths from undifferentiated inner cell mass cells when embryos are plated onto inactivated mouse embryonic feeder layers.

Characterization of stem-like cells in mucoepidermoid tracheal paediatric tumor.

Stem cells contribute to regeneration of tissues and organs. Cells with stem cell-like properties have been identified in tumors from a variety of origins, but to our knowledge there are yet no reports on tumor-related stem cells in the human upper respiratory tract. In the present study, we show that a tracheal mucoepidermoid tumor biopsy obtained from a 6 year-old patient contained a subpopulation of cells with morphology, clonogenicity and surface markers that overlapped with bone marrow mesenchymal stromal cells (BM-MSCs). These cells, designated as MEi (mesenchymal stem cell-like mucoepidermoid tumor) cells, could be differentiated towards mesenchymal lineages both with and without induction, and formed spheroids in vitro. The MEi cells shared several multipotent characteristics with BM-MSCs. However, they displayed differences to BM-MSCs in growth kinectics and gene expression profiles relating to cancer pathways and tube development. Despite this, the MEi cells did not possess in vivo tumor-initiating capacity, as proven by the absence of growth in situ after localized injection in immunocompromised mice. Our results provide an initial characterization of benign tracheal cancer-derived niche cells. We believe that this report could be of importance to further understand tracheal cancer initiation and progression as well as therapeutic development.

Epiblast Cell Number and Primary Embryonic Stem Cell Colony Generation Are Increased by Culture of Cleavage Stage Embryos in Insulin

Abstract Human embryos for hESC derivation are often donated at the cleavage stage and of reduced quality. Poor quality embryos have lower efficiency for hESC derivation. However, cleavage stage mouse embryos develop into higher quality expanded blastocysts if they are cultured with insulin, suggesting that this approach could be used to improve hESC derivation from poor quality cleavage stage embryos. The present study used a mouse model to examine this approach. In particular we examined the effect of insulin on the number of epiblast cells in blastocysts on days 4, 5 and 6 using Oct4 and Nanog co-expression. Second we examined the effect of insulin on the frequency with which outgrowths can be derived from these. Finally, we tested whether prior culture in the presence of insulin results in blastocysts with increased capacity to generate ESC colonies. Culture of cleavage stage embryos with insulin increased the number of Oct4 and Nanog positive cells in blastocysts at all time points examined. Prior culture with insulin had no effect on outgrowths generated from blastocysts plated on days 4 or 5. However, insulin treatment of blastocysts plated on day 6 resulted in increased numbers of outgrowths with larger epiblasts compared with controls. 13% of insulin treated day 6 blastocysts produced primary ESC colonies compared with 6% of controls. In conclusion, treatment with insulin can improve epiblast cell number in mice leading to an increase with which primary ESC colonies can be generated and may improve hESC isolation from reduced quality embryos donated at the cleavage stage.

Development of an Improved Porcine Embryo Culture Medium for Cloning, Transgenesis and Embryonic Stem Cell Isolation

Work in our laboratory for more than two decades has focussed on the production of genetically modified pigs for xeno transplantion research. More recent work has focussed on the isolation of porcine embryonic stem cells to facilitate this as well as and other research applications. Central to this research has been the production of in vitro Produced (IVP) embryos. These embryos are produced using a twostep culture system based on NCSU23. This culture system which was developed by modifying energy substrate availability and concentrations and by adding non-essential and essential amino acids in a sequential manner. As a result of this work we have developed a culture system that better suits the changing metabolic needs of the pig embryo and produces embryos with relatively high developmental competence compared to the original formulation. These embryos can be used for a range of research applications including the isolation of embryonic stem cells.

Targeted insertion of an anti-CD2 monoclonal antibody transgene into the GGTA1 locus in pigs using Fok I-dCas9

Xenotransplantation from pigs has been advocated as a solution to the perennial shortage of donated human organs and tissues. CRISPR/Cas9 has facilitated the silencing of genes in donor pigs that contribute to xenograft rejection. However, the generation of modified pigs using second-generation nucleases with much lower off-target mutation rates than Cas9, such as FokI-dCas9, has not been reported. Furthermore, there have been no reports on the use of CRISPR to knock protective transgenes into detrimental porcine genes. In this study, we used FokI-dCas9 with two guide RNAs to integrate a 7.1 kilobase pair transgene into exon 9 of the GGTA1 gene in porcine fetal fibroblasts. The modified cells lacked expression of the αGal xenoantigen, and secreted an anti-CD2 monoclonal antibody encoded by the transgene. PCR and sequencing revealed precise integration of the transgene into one allele of GGTA1, and a small deletion in the second allele. The cells were used for somatic cell nuclear transfer to generate healthy male knock-in piglets, which did not express αGal and which contained anti-CD2 in their serum. We have therefore developed a versatile high-fidelity system for knocking transgenes into the pig genome for xenotransplantation purposes.