Clinical animal cytogenetics development began in the 1960’s, almost at the same time as human cytogenetics. However, the development of the two disciplines has been very different during the last four decades. Clinical animal cytogenetics reached its ‘Golden Age’ at the end of the 1980’s. The majority of the laboratories, as well as the main screening programs in farm animal species, presented in this review, were implemented during that period, under the guidance of some historical leaders, the first of whom was Ingemar Gustavsson. Over the past 40 years, hundreds of scientific publications reporting original chromosomal abnormalities generally associated with clinical disorders (mainly fertility impairment) have been published. Since the 1980’s, the number of scientists involved in clinical animal cytogenetics has drastically decreased for different reasons and the activities in that field are now concentrated in only a few laboratories (10 to 15, mainly in Europe), some of which have become highly specialized. Currently between 8,000 and 10,000 chromosomal analyses are carried out each year worldwide, mainly in cattle, pigs, and horses. About half of these analyses are performed in one French laboratory. Accurate estimates of the prevalence of chromosomal abnormalities in some populations are now available. For instance, one phenotypically normal pig in 200 controlled in France carries a structural chromosomal rearrangement. The frequency of the widespread 1;29 Robertsonian translocation in cattle has greatly decreased in most countries, but remains rather high in certain breeds (up to 20–25% in large beef cattle populations, even higher in some local breeds). The continuation, and in some instances the development of the chromosomal screening programs in farm animal populations allowed the implementation of new and original scientific projects, aimed at exploring some basic questions in the fields of chromosome and/or cell biology, thanks to easier access to interesting biological materials (germ cells, gametes, embryos ...).

Background Human induced pluripotent stem cells (hiPSCs) hold enormous promise in accelerating breakthroughs in understanding human development, drug screening, disease modeling, and cell and gene therapies. Their potential, however, has been bottlenecked in a mostly laboratory setting due to bioprocess challenges in the scale-up of large quantities of high-quality cells for clinical and manufacturing purposes. While several studies have investigated the production of hiPSCs in bioreactors, the use of conventional horizontal-impeller, paddle, and rocking-wave mixing mechanisms have demonstrated unfavorable hydrodynamic environments for hiPSC growth and quality maintenance. This study focused on using computational fluid dynamics (CFD) modeling to aid in characterizing and optimizing the use of vertical-wheel bioreactors for hiPSC production. Methods The vertical-wheel bioreactor was modeled with CFD simulation software Fluent at agitation rates between 20 and 100 rpm. These models produced fluid flow patterns that mapped out a hydrodynamic environment to guide in the development of hiPSC inoculation and in-vessel aggregate dissociation protocols. The effect of single-cell inoculation on aggregate formation and growth was tested at select CFD-modeled agitation rates and feeding regimes in the vertical-wheel bioreactor. An in-vessel dissociation protocol was developed through the testing of various proteolytic enzymes and agitation exposure times. Results CFD modeling demonstrated the unique flow pattern and homogeneous distribution of hydrodynamic forces produced in the vertical-wheel bioreactor, making it the opportune environment for systematic bioprocess optimization of hiPSC expansion. We developed a scalable, single-cell inoculation protocol for the culture of hiPSCs as aggregates in vertical-wheel bioreactors, achieving over 30-fold expansion in 6 days without sacrificing cell quality. We have also provided the first published protocol for in-vessel hiPSC aggregate dissociation, permitting the entire bioreactor volume to be harvested into single cells for serial passaging into larger scale reactors. Importantly, the cells harvested and re-inoculated into scaled-up vertical-wheel bioreactors not only maintained consistent growth kinetics, they maintained a normal karyotype and pluripotent characterization and function. Conclusions Taken together, these protocols provide a feasible solution for the culture of high-quality hiPSCs at a clinical and manufacturing scale by overcoming some of the major documented bioprocess bottlenecks.

Six local chicken breeds are registered in Hungary and are regarded as Hungarian national treasures: Hungarian White, Yellow and Speckled, and Transylvanian Naked Neck White, Black and Speckled. Three Hungarian academic institutes have maintained these genetic resources for more than 30 years. The Hungarian Yellow, the Hungarian Speckled and the Transylvanian Naked Neck Speckled breeds were kept as duplicates in two separate subpopulations since time of formation of conservation flocks at different institutes. In this study, we investigated genetic diversity of these nine Hungarian chicken populations using 29 microsatellite markers. We assessed degree of polymorphism and relationships within and between Hungarian breeds on the basis of molecular markers, and compared the Hungarian chicken populations with commercial lines and European local breeds. In total, 168 alleles were observed in the nine Hungarian populations. The F(ST) estimate indicated that about 22% of the total variation originated from variation between the Hungarian breeds. Clustering using structure software showed clear separation between the Hungarian populations. The most frequent solutions were found at K = 5 and K = 6, respectively, classifying the Transylvanian Naked Neck breeds as a separate group of populations. To identify genetic resources unique to Hungary, marker estimated kinships were estimated and a safe set analysis was performed. We show that the contribution of all Hungarian breeds together to the total diversity of a given set of populations was lower when added to the commercial lines than when added to the European set of breeds.

In this study, we assessed the maternal origin of six Hungarian indigenous chicken breeds using mitochondrial DNA information. Sequences of Hungarian chickens were compared with the D-loop chicken sequences annotated in the GenBank and to nine previously described reference haplotypes representing the main haplogroups of chicken. The first 530 bases of the D-loop region were sequenced in 74 chickens of nine populations. Eleven haplotypes (HIC1-HIC11) were observed from 17 variable sites. Three sequences (HIC3,HIC8 and HIC9) of our chickens were found as unique to Hungary when searched against the NCBI GenBank database. Hungarian domestic chicken mtDNA sequences could be assigned into three clades and probably two maternal lineages. Results indicated that 86%of the Hungarian haplotypes are related to the reference sequence that likely originated from the Indian subcontinent, while the minor part of our sequences presumably derive from South East Asia, China and Japan.

In this study, we used genetic-based approaches to estimate population size and structure of Eurasian otter along the Drava River in Hungary, and compared these results to traditional survey-based methods. The relative spraint density of otter was estimated based on the number of fresh (D f ) and total number (D t ) of spraints collected on standard routes over a 2-year period. Nine microsatellite loci were screened, generating 17 individual otter genotypes composed of 45 different alleles. The expected heterozygosity ranged from 0.53 to 0.89 and observed heterozygosity from 0.25 to 0.92. The mean density (D g ) estimated over six different sites was 0.17 individuals per km of shoreline. A close correlation was found between the number of genotypes and spraint counts along a standard route (fresh spraints: r P ¼ 0.85, Po0.01; total spraints r P ¼ 0.76, Po0.05). All genotypes found within the 50 km-long study area were closely related (D m ranged between 0.08 and 0.21).

Heat shock transcription factor, Y-linked (HSFY) is a member of the heat shock transcriptional factor (HSF) family that is found in multiple copies on the Y chromosome and conserved in a number of species. Its function still remains unknown but in humans it is thought to play a role in spermatogenesis. Through real time polymerase chain reaction (PCR) analyses we determined that the HSFY family is largely expanded in cattle (∼70 copies) compared with human (2 functional copies, 4 HSFY-similar copies). Unexpectedly, we found that it does not vary among individual bulls as a copy number variant (CNV). Using fluorescence in situ hybridization (FISH) we found that the copies are dispersed along the long arm of the Y chromosome (Yq). HSFY expression in cattle appears restricted to the testis and its mRNA correlates positively with mRNA markers of spermatogonial and spermatocyte cells (UCHL1 and TRPC2, respectively) which suggests that HSFY is expressed (at least in part) in early germ cells.

Human induced pluripotent stem cells (hiPSCs) have generated a great deal of attention owing to their capacity for self‐renewal and differentiation into the three germ layers of the body. Their discovery has facilitated a new era in biomedicine for understanding human development, drug screening, disease modeling, and cell therapy while reducing ethical issues and risks of immune rejection associated with traditional embryonic stem cells. Bioreactor‐based processes have been the method of choice for the efficient expansion and differentiation of stem cells in controlled environments. Current protocols for the expansion of hiPSCs use horizontal impeller, paddle, or rocking wave mixing method bioreactors which require large static cell culture starting populations and achieve only moderate cell fold increases. This study focused on optimizing inoculation, agitation, oxygen, and nutrient availability for the culture of hiPSCs as aggregates in single‐use, low‐shear, vertical‐wheel bioreactors. Under optimized conditions, we achieved an expansion of more than 30‐fold in 6 days using a small starting population of cells and minimal media resources throughout. Importantly, we showed that that this optimized bioreactor expansion protocol could be replicated over four serial passages resulting in a cumulative cell expansion of 1.06E6‐fold in 28 days. Cells from the final day of the serial passage were of high quality, maintaining a normal karyotype, pluripotent marker staining, and the ability to form teratomas in vivo. These findings demonstrate that a vertical‐wheel bioreactor‐based bioprocess can provide optimal conditions for efficient, rapid generation of high‐quality hiPSCs to meet the demands for clinical manufacturing of therapeutic cell products.

Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer and often is not detected until late stages when cancer cells transcoelomically metastasize to the abdomen and typically become resistant to therapy resulting in very low survival rates. We utilize an orthotopic, syngeneic mouse model to study late stage disease and have discovered that the tumor cells within the abdominal ascites are irreversibly re-programmed, with an increased tumorigenicity and resistance to apoptosis. The goal of this study was to characterize the reprogramming that occurred in the aggressive ascites-derived cells (28-2 cells) compared to the original cell line used for tumor induction (ID8 cells). Microarray experiments showed that the majority of genes upregulated in the 28-2 cells belonged to the mevalonate pathway, which is involved in cholesterol biosynthesis, protein prenylation, and activation of small GTPases. Upregulation of mevalonate appeared to be associated with the acquisition of a p53 mutation in the ascites-derived cells. Treatment with simvastatin to inhibit HMG CoA reductase, the rate limiting enzyme of this pathway, induced apoptosis in the 28-2 cell line. Rescue experiments revealed that mevalonate, but not cholesterol, could inhibit the simvastatin-mediated effects. In vivo, daily intraperitoneal simvastatin treatment significantly regressed advanced stage disease and induced death of metastatic tumor cells. These data suggest that ovarian cancer cells become reprogrammed, with genetic mutations, and upregulation of the mevalonate pathway, which facilitates the development of advanced stage disease. The use of statins to inhibit HMGCR may provide novel therapeutic opportunities for the treatment of advanced stage EOC.