Stem cells and embryology – University of Copenhagen

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Stem cells and embryology

Our group entails diverse projects encompassing the generation and study of induced pluripotent stem cells (iPSC) and their differentiated derivatives from humans, dogs and pigs. We also have a strong background in the study of oocytes and in vitro produced and somatic cell nuclear transfer embryos derived from large domestic animals. Our lab caters for performing varying histological, molecular and imaging technologies. We have two GMO Class 1 facilities, diverse molecular labs and are equipped with a transmission electron microscope, confocal and time-lapse microscope.

Development of in vitro models of neurodegenerative diseases based on induced pluripotent stem cells (iPSCs)

Disorders of the nervous system pose an exponentially growing health care challenge for all Western countries including Denmark. Future treatments of incurable neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease need to be tailored to patient’s needs in order to obtain an optimal effect.

The main objectives of our research group are to (1) refine patient-specific iPSC-derived models for disorders such as Alzheimer’s disease and Parkinson’s disease for studying molecular pathology and (2) establish patient-specific in vitro screening platforms for neurodegeneration. The hope is to use these patient-specific stem cells, derived from simple skin biopsies, to provide better diagnostics and treatment, which are urgently required for almost all neurological disorders.

We are the home of the stem cell center BrainStem (see http://brainstem.dk), funded by Innovation Fund Denmark, a European Union-supported project (STEMMAD) as well as the Indo-Danish project NeuroStem, also supported by Innovation Fund Denmark. These projects aim at generating patient-specific iPSC lines from patients with neurodegenerative diseases as e.g. Alzheimer’s disease and Parkinson's disease, which upon differentiation into neural cells can be used as in vitro cell models that will ultimately allow for investigation of molecular disease mechanisms, discovery of novel drug targets and for development of earlier diagnostics and customized treatments. NeuroStem has a particular focus on the discovery of RNA-mediated regulation in neurodegeneration.

Over the last few years, we have implemented a very stable and consistently working non-integrative technique to establish iPSCs from fibroblasts obtained from patients suffering from neurodegenerative diseases. These iPSCs are further differentiated into disease relevant neural subtypes and assessed for characteristic pathological phenotypes. We are currently studying the pathological pathways in our iPSC-derived neurodegenerative models including studies on neuronal function assessed by Ca2+ signalling, live cell imaging to analyse dendritic spine formation and receptor turnover on synapses, and transmission electron microscopy to investigate defects in endosomes, autophagy and mitochondria.  We hope in the future to perform high throughout chemical compound screenings on these cells, in order to identify new targets that could be used for future drug development for these diseases.

In a novel project, recently granted by the  Danish Agency for Science, Technology and Innovation, we will develop the dog as a refined model for Alzheimer’s disease. Our overall hypothesis is that that the domestic dog with naturally occurring dementia, based on their close phylogenetic, physiological and social similarity to humans, offers significant advantages to existing experimental animal models for investigating early stages of human Alzheimer’s disease (AD). Building on our previous promising results it is our objectives by advanced technologies to take the demented dog model from “bedside to bench”: (1) At the clinical level by investigating the clinical and pathological similarities between dogs spontaneously affected by canine cognitive dysfunction (CCD) and humans suffering from AD. (2) At the cellular and molecular level by finding commonalities between disease phenotype in stem cell-derived neurons from CCD dogs and human AD patients. As a future perspective, the project is expected to set the stage for (1) using stem cell-derived neurons from CCD dogs and human AD patients for drug discovery and (2) for further in vivo validation of drug candidates in dogs affected by CCD. 

Porcine pluripotent stem cells

The pig is an attractive alternative animal model for studies of potentials and risks of future iPSC-based therapy. Hence, several groups have produced porcine induced pluripotent stem cells (iPSCs), but it has since become apparent that porcine iPSCs often show persistent expression of the reprogramming factors and lack of endogenous pluripotency gene activation. We have used a non-integrative vector based reprogramming approach for porcine iPSC reprogramming. However, persistence of the exogenous genes was detected at passage 10, but longer culture, followed by picking of individual clones and expansion of these, lead to loss of exogenous gene detection. Our study shows to our knowledge, for the first time, the generation of transgene-free porcine iPSCs. These safer porcine iPSCs would be ideal cell lines for the generation of porcine in vitro cell models and potentially used in vivo or for exploring the potentials and safety of human iPSC-based therapy.

Further improvement in the production efficiency of porcine iPSC could be achieved by selecting the most appropriate starting cell type for reprogramming. Although iPSC can be produced from any somatic cell type of the body, there are clear differences in reprogramming efficiencies, which is highly dependent on the original cell population. This has also been shown in the pig, as only one cell type used (namely bone marrow-derived mesenchymal stem cells) has resulted in production of porcine iPSC that could contribute to the germ line. By screening of porcine fibroblast cell lines derived from three different pig breeds, we observed a minor population of stage-specific embryonic antigen 1(SSEA-1) cells. Reprogramming studies revealed that SSEA-1+ sorted cells were in fact more highly amenable to reprogramming. This may help in selecting more amenable cell types for reprogramming to enhance the process and to generate more robust iPSCs in the future.

Currently, it is a big hurdle to translate scientific observations from mouse models to humans, as human phenotypes are lacking the mouse or drug responses in the mouse are not reproducible in human clinical trials. This makes it obvious that there is a need for more relevant animal models, which more closely resemble humans in regards to genetics, physiology and anatomy such as the pig. Hence, the generation of porcine iPSCs has long been considered as an ultimate goal in order to establish this species as a faithful model of iPSC-based cell therapy. Nevertheless, the attempts to establish stable porcine iPSC have been hampered by insufficient activation of endogenous pluripotency genes, lack of silencing of integrated pluripotency transgenes and suboptimal knowledge of the cell culture conditions, which also have prevented isolation of porcine embryonic stem cells (pESC).

The golden standard for demonstrating pluripotency is the production of chimeric embryos developing into germ line transmitting offspring. In order to test the capacity of porcine iPSC to contribute to chimeric embryo formation, we have generated two porcine iPSC lines, which have been generated using four porcine pluripotency genes under control of a doxycycline-dependent promoter, and which have been maintained either with 2i LIF or 2i FGF. There appeared to be a small advantage of the 2i LIF cells in the survival after blastocyst injection, but this was doxycycline dependent and thereby transgene dependent. Similarly, teratomas could only be obtained when doxycycline was fed to the NOD/SCID mice. Upon transfer of chimeric embryos to recipient sows, the 2i LIF iPSC-derivatives could only be detected in the embryonic membranes during early pregnancy.

In vitro fertilization in cattle

Reproductive technologies are important tools in cattle breeding with Artificial Insemination (AI) used for more than 50 years and Embryo Transfer (ET) used since the mid-1980s. More recently, ET of in vitro produced (IVP) embryos derived from ultrasound-guided collection of oocytes (Ovum Pick-Up, OPU) from superior donor cows has been introduced. This technique is used increasingly worldwide, but there are needs for improved understanding of the biology as well as the associated quantitative genetics in order to exploit the full benefits. The aims of the GIFT (Genomic Improvement of Fertilization traits in Danish and Brazilian Cattle; principal investigator: Professor Haja Kadarmideen) project are to form a bridge between embryo technology and genetics/genomics in order to address complex biological mechanisms for OPU-IVP traits as well as higher pregnancy rates, find key genes and genetic markers, and develop genomic selection programs for identification of superior donors and recipients. The GIFT partners in Brazil and Denmark represent in collaboration a unique set of competences, which synergistically support each other in strong basic genomics and embryological experiences, extensive practical use of the techniques, access to both beef and dairy cattle and two breeds (Bos Taurus and Bos Indicus), as well as two environments (tropical and temperate climate). Hence, the GIFT project allows for a novel assessment of biological and genetic dimensions of the OPU-IVP technology in a contemporary breeding context. Overall, the GIFT project combines two practical embryo and genomic technologies to make a direct contribution to quality and quantity of meat and milk produced from cattle and used for human consumption.