Pablo Bosch

Isolation, Characterization, Gene Modification, and Nuclear Reprogramming of Porcine Mesenchymal Stem Cells

Abstract Bone marrow mesenchymal stem cells (MSCs) are adult pluripotent cells that are considered to be an important resource for human cell-based therapies. Understanding the clinical potential of MSCs may require their use in preclinical large-animal models, such as pigs. The objectives of the present study were 1) to establish porcine MSC (pMSC) cultures; 2) to optimize in vitro pMSC culture conditions, 3) to investigate whether pMSCs are amenable to genetic manipulation, and 4) to determine pMSC reprogramming potential using somatic cell nuclear transfer (SCNT). The pMSCs isolated from bone marrow grew, attached to plastic with a fibroblast-like morphology, and expressed the mesenchymal surface marker THY1 but not the hematopoietic marker ITGAM. Furthermore, pMSCs underwent lipogenic, chondrogenic, and osteogenic differentiation when exposed to specific inducing conditions. The pMSCs grew well in a variety of media, and proliferative capacity was enhanced by culture under low oxygen atmosphere. Transient transduction of pMSCs and isogenic skin fibroblasts (SFs) with a human adenovirus carrying the gene for green fluorescent protein (GFP; Ad5-F35eGFP) resulted in more pMSCs expressing GFP compared with SFs. Cell lines with stable genetic modifications and extended expression of transgene were obtained when pMSCs were transfected with a plasmid containing the GFP gene. Infection of pMSC and SF cell lines by an adeno-associated virus resulted in approximately 12% transgenic cells, which formed transgenic clonal lines after propagation as single cells. The pMSCs can be expanded in vitro and used as nuclear donors to produce SCNT embryos. Thus, pMSCs are an attractive cell type for large-animal autologous and allogenic cell therapy models and for SCNT transgenesis.

Development of antral follicles in cryopreserved cat ovarian tissue transplanted to immunodeficient mice

Ovarian cortex cryopreservation and xenotransplantation into immunodeficient mice represents a potential means for female germplasm conservation and an immediate model for investigation of folliculogenesis. The objectives of this study were to: (1) assess follicle survival after cryopreservation and transplantation of cat ovarian tissue into non-obese diabetic severely combined immunodeficient (NOD SCID) mice; and (2) evaluate the effects of gonadotropin treatments on follicular development in the transplanted tissue. Slices from the cat ovarian cortex were frozen and after thawing, transplanted under each kidney capsule of castrated male NOD SCID mice (eight xenografts in four mice). Sixty-two days after surgery, mice were randomly assigned (two per group) to gonadotropin-treated (eCG and hCG 88 h later) or control (saline-treated) groups. Twenty-four hours after the last injection, ovarian tissue was recovered and processed for histology. Fresh ovarian tissue from the same original source was similarly processed. Follicles were counted, measured, and classified as primordial, primary, secondary, or antral. Immunoreactive proliferating cell nuclear antigen (PCNA) stain was used to assess follicle viability. Microscopic examination revealed no evidence of necrosis or fibrosis. The grafts were well-vascularized, with follicles at all stages of development. Numbers of follicles in the transplanted tissue were markedly reduced compared to fresh tissue, with approximately 10% of follicles surviving freezing and transplantation procedures. Growing follicles positive for PCNA were found in all xenografts. Gonadotropin treatment did not alter the proportion of resting to growing follicles or mean follicle diameter by comparison with controls from untreated mice. By contrast, luteinization, but not ovulation, of antral follicles was observed only in grafts from treated mice. In summary, frozen-thawed cat ovarian cortex tissue not only survived xenotransplantation, it also contained follicles able to grow to antral stages. Exogenous gonadotropin treatment in this model resulted in luteinization of antral follicles but enhancement of follicular growth and ovulation did not occur.

Effect of leukemia inhibitory factor on bovine embryos produced in vitro under chemically defined conditions

The objective of these experiments was to assess putative embryotrophic effects of leukemia inhibitory factor (LIF) on bovine preimplantation development in chemically defined media. Recombinant human LIF was added to embryo culture media at a concentration of 100 ng/ml. When added for culture of morulae LIF had no positive effect on the proportion of embryos reaching the blastocyst stage. However, LIF significantly reduced development to the blastocyst stage when added for culture of 4-cell stage embryos (P<0.05). In contrast, a positive effect was found for progression of blastocyst development. In vitro blastocyst hatching rates were significantly improved in the presence of LIF (P<0.02). Number of total cells and of inner cell mass (ICM) cells were increased in LIF-treated blastocysts. In vitro survival of frozen-thawed blastocysts was not improved by adding LIF to morula stage embryos before cryopreservation. The pregnancy rate after direct transfer of cryopreserved LIF-treated embryos was not different from that for untreated control embryos. Data indicate that addition of LIF has no major beneficial effect on bovine embryos produced in these chemically defined conditions.

One-step Multiplex Transgenesis via Sleeping Beauty Transposition in Cattle

Genetically modified cattle are important for developing new biomedical models and for an improved understanding of the pathophysiology of zoonotic diseases. However, genome editing and genetic engineering based on somatic cell nuclear transfer suffer from a low overall efficiency. Here, we established a highly efficient one-step multiplex gene transfer system into the bovine genome.

Adenoviral Transduction of Mesenchymal Stem Cells

Intact or genetically manipulated mesnechymal stem cells (MSCs) are being considered an important cell source for developing human cell-based therapeutic approaches. For applications in which transient, high-level expression of the transgene is necessary, adenovirus vectors have become increasingly popular gene-transfer vehicles. However, host range and cell-type tropism restrict the use of specific adenovectors, sometimes necessitating the lengthy development of vectors with appropriate cell specificity. Here, we present a versatile and inexpensive porcine MSC transduction procedure that can also be used on other cell types from various species, including human that are otherwise refractory to adenovirus infection.

203 Genome Modifications by Sleeping Beauty Transposition and CRISPR/Cas9 to Improve Cow Milk Composition for Human Consumption

Genome manipulation of cattle represents a powerful tool to increase the nutritional value and reduce allergenicity of cow milk for human consumption. This could be accomplished by improving the amount of polyunsaturated fatty acids (ω-3 and ω-6) and simultaneously abolishing β-lactoglobulin (BLG), a potent allergen for predisposed humans. The aim of this study was to introduce the sequence for a desaturase construct (mFAT-2, from C. elegans), which is able to catalyse the synthesis of ω-3 and ω-6 fatty acids, into the bovine genome by Sleeping Beauty (SB) transposition, and simultaneously knocking out the bovine β-lactoglubulin gene using CRISPR/Cas9 system. The sgRNA (AAGTGCCTCCTGCTTGCCC) targeted to BLG exon 1 was synthesised as an oligo linker and cloned into the px459-Cas9. The mutation activity of the designed sgRNA at the target locus was determined by T7 endonuclease assay I (T7EI) mismatch detection assay. Briefly, bovine fetal fibroblasts (BFF) were seeded at 0.5 × 105 cells per well of a 24-well plate in triplicate, when the cells reached 80% confluence (12–24 h), cultures were transfected with 1 μg of px459-Cas9::BLG plasmid co-expressing Cas9 and sgRNA using polyethylenimine reagent (PEI; 3 ng μL−1). After 3 days of puromycin selection, genomic DNA from transfected cells were extracted and the sequence of interest was PCR-amplified and digested by T7EI restriction enzyme. Digestion products showed a mutation efficiency at the target locus of 29%. Subsequently, we chemically cotransfected 0.5 × 105 BFF with 0.5 μg of knockout vector (px459-Cas9::BLG) and 0.5 μg of SB plasmids (carrying mFAT-2 cDNA for mammary gland-specific expression) using 3 ng μL−1 PEI in triplicate. At 48 h post-transfection, cell cultures were subjected to 3 days of puromycin and 21 days of neomycin selection. PCR analysis of antibiotic resistant colonies revealed the presence of mFAT-2 transgene in almost 70% of the analysed cells lines. Genotyping of BLG exon 1 was performed by direct sequencing of PCR amplicons using primers flanking the target site. Despite the appreciable gene mutation activity of the sgRNA sequence previously determined by T7EI assay (29%), none of the cell lines analysed showed modification in the BLG target locus. We speculate that the SB vector might have disrupted the activity of targeting vector. We are currently performing additional experiments to accomplish gene addition (mFAT) and gene knockout (BGL) in one step using these highly efficient and precise transgenic tools. Genetically modified cells will be used as nuclear donor to produce transgenic cows by somatic cells nuclear transfer. The financial support of CONICET, UNRC and FONCYT is gratefully acknowledged.

Attenuation effects of prenatal stress by early postnatal stimulation in different research models

The prenatal and early postnatal life stages are both dynamic and vulnerable phases during mammalian development. Exposure to adverse factors that interfere with this critical sequence of events places the exposed individual to a higher risk of developing various disorders in adult life.1–3 For instance, high blood glucocorticoids concentration in pregnant stressed females crossing the placenta and the fetal blood-brain barriers,4 can affect brain development, birth weight and HPA axis function in offspring.5

Stress versus immunity

Stress can be defined as a real or supposed threat to physical or psychological integrity of an individual, resulting in a physiological and /or behavioral response.1 The degree of damage caused by the stress depends on the nature, intensity and duration of the stimuli as well as the stage of gestation at which the stressor is applied.2 Dhabhar & McEwen3 showed that the activation of the physiologic stress response systems can also enhance immune function as evidenced by increase in allergic contact sensitivity or delayed-type hypersensitivity. In contrast, acute stress has no effect on the course of irritant contact sensitivity, an immune reaction that does not involve an Ag-specific memory response. Herbert & Cohen4 suggested that objective stressful events leas to larger immune changes than subjective self-reports of stress and that interpersonal events are related to different immune outcomes than non-social events.

Multiplex transgenesis in cattle via the sleeping beauty transposon system

Program and Abstracts of the 13th Transgenic Technology Meeting (TT2016) Clarion Congress Hotel, Prague, Czech Republic, 20–23 March 2016 The TT2016 meeting is hosted by: the Czech Centre for Phenogenomics (CCP), BIOCEV, Prumyslova 595, 25242, Vestec, Czech Republic Biotechnology and Biomedicine Center of the Academy of Sciences and Charles University in Vestec, Prumyslova 595, 252 42, Vestec, Czech Republic Institute of Molecular Genetics of the ASCR, v. v. i. Vı́deňská 1083, 142 20 Prague 4, Czech Republic 123 Transgenic Res (2016) 25:195–270 Springer International Publishing Switzerland 2016 DOI 10.1007/s11248-016-9936-6