Final Report Summary - ANISTEM (Induced pluripotent stem cells in rabbit, pig)
Summary description of the project objectives
The ANISTEM project was aimed at creating a 4-years training and transfer of knowledge program between a Hungarian SME and a Dutch and a Hungarian University in the field of stem cell research in mammalians (rabbit, pig), and to open opportunities for a mutually beneficial long term partnership of veterinary and medical research and for applications between the industrial and academic participants.
The scientific and industrial aim of ANISTEM have been to generate new comparative information on the generation, maintenance and characteristics of induced Pluripotent Stem (iPS) cells in rabbit and pig and on their differentiation capacity.
Description of the work performed since the beginning of the project
The five most important pluripotency markers from rabbit (OCT4, SOX2, KLF4, cMYC and NANOG) were identified and isolated. For this step phylogenetic analysis were performed, and based on the sequence analysis degenerative primers were used to isolate the cDNA of the pluripotency markers. To generate rabbit induced pluripotent stem cells (rbiPSC) two different reprogramming methods were used: lentiviral and protein transduction. Two different lentiviral constructs were used; one contained the mouse pluripotency markers (OSKM) and the eGFP as reporter protein under the control of the EF1alpha promoter. In the other lentiviral construct the codon optimized human pluripotency factors were present (OKSM) and contained the dTomato as reporter protein under the control of a retroviral promoter, which theoretically silenced. By using the human factors containing lentiviral construct, two rbiPSC lines were generated and characterized for their pluripotency marker expression pattern and their in vitro differentiation ability towards the three germ layer lineages. The generation of rbIPSCs by protein transduction was also successful as some of the obtained colonies formed teratomas containing cell types from all three germ layers after injection into nude mice.
Porcine iPS cells have been obtained via collaboration with the Hyttel-group at Copenhagen University. Four different cell lines were further examined. The differences among these cell lines are due to the method of generation, in particular whether the cells where generated in the presence of leukemia inhibitory factor (LIF) or fibroblast growth factors (FGF), and two types of reporter construct: Venus and mCherry. The cells have been cultured under various conditions with various signalling pathways inhibitors and the cells have been characterized (gene and protein expression, differentiation capacity). The cell lines have been tested further for their differentiation capacity into cardiac, smooth muscle and other directions. Partial success have been achieved with the pluripotent cell types, however only limited differentiation potential have been achieved complicated by a tendency to loose pluripotency.
Description of the main results achieved
The consortium identified and isolated the 5 rabbit pluripotency markers: OCT4, SOX2, KLF4, cMYC and NANOG; and successfully generated several rabbit induced pluripotent stem cell lines (rbiPSC) and characterized them for pluripotency marker expression pattern by IHC and verified their ability to differentiate in vitro towards the three germ layer lineages. It was possible to obtain stable and good quality rbiPSC lines.
The acquired porcine iPS cells have been cultured under various conditions. RNA and protein expression of pluripotency genes, proliferation and differentiation capacity has been examined. In addition, it has been examined whether different culture conditions facilitated the culture of the cells in the absence of the exogenous pluripotency factors, but only limited success have been achieved.
The expected final results and their potential impact and use
The main impact of the project is expected to be in integrating efforts in stem cell biology, biomedical models and cell therapy between industry and academia for long term collaboration. Better stem cell differentiation protocols in mammalians will potentially allow for validation and development of iPS cells as highly desirable alternatives for live animals for drug and toxicity testing, disease modeling and research on regenerative medicine.
AniStem has allowed the consortium members to develop beyond the current state-of-the-art novel methods for producing iPS cells of rabbit and pig origin. This potentially will create a platform for future research on drugs to delay or even stop disease onset in humans and animals.
The ANISTEM project was aimed at creating a 4-years training and transfer of knowledge program between a Hungarian SME and a Dutch and a Hungarian University in the field of stem cell research in mammalians (rabbit, pig), and to open opportunities for a mutually beneficial long term partnership of veterinary and medical research and for applications between the industrial and academic participants.
The scientific and industrial aim of ANISTEM have been to generate new comparative information on the generation, maintenance and characteristics of induced Pluripotent Stem (iPS) cells in rabbit and pig and on their differentiation capacity.
Description of the work performed since the beginning of the project
The five most important pluripotency markers from rabbit (OCT4, SOX2, KLF4, cMYC and NANOG) were identified and isolated. For this step phylogenetic analysis were performed, and based on the sequence analysis degenerative primers were used to isolate the cDNA of the pluripotency markers. To generate rabbit induced pluripotent stem cells (rbiPSC) two different reprogramming methods were used: lentiviral and protein transduction. Two different lentiviral constructs were used; one contained the mouse pluripotency markers (OSKM) and the eGFP as reporter protein under the control of the EF1alpha promoter. In the other lentiviral construct the codon optimized human pluripotency factors were present (OKSM) and contained the dTomato as reporter protein under the control of a retroviral promoter, which theoretically silenced. By using the human factors containing lentiviral construct, two rbiPSC lines were generated and characterized for their pluripotency marker expression pattern and their in vitro differentiation ability towards the three germ layer lineages. The generation of rbIPSCs by protein transduction was also successful as some of the obtained colonies formed teratomas containing cell types from all three germ layers after injection into nude mice.
Porcine iPS cells have been obtained via collaboration with the Hyttel-group at Copenhagen University. Four different cell lines were further examined. The differences among these cell lines are due to the method of generation, in particular whether the cells where generated in the presence of leukemia inhibitory factor (LIF) or fibroblast growth factors (FGF), and two types of reporter construct: Venus and mCherry. The cells have been cultured under various conditions with various signalling pathways inhibitors and the cells have been characterized (gene and protein expression, differentiation capacity). The cell lines have been tested further for their differentiation capacity into cardiac, smooth muscle and other directions. Partial success have been achieved with the pluripotent cell types, however only limited differentiation potential have been achieved complicated by a tendency to loose pluripotency.
Description of the main results achieved
The consortium identified and isolated the 5 rabbit pluripotency markers: OCT4, SOX2, KLF4, cMYC and NANOG; and successfully generated several rabbit induced pluripotent stem cell lines (rbiPSC) and characterized them for pluripotency marker expression pattern by IHC and verified their ability to differentiate in vitro towards the three germ layer lineages. It was possible to obtain stable and good quality rbiPSC lines.
The acquired porcine iPS cells have been cultured under various conditions. RNA and protein expression of pluripotency genes, proliferation and differentiation capacity has been examined. In addition, it has been examined whether different culture conditions facilitated the culture of the cells in the absence of the exogenous pluripotency factors, but only limited success have been achieved.
The expected final results and their potential impact and use
The main impact of the project is expected to be in integrating efforts in stem cell biology, biomedical models and cell therapy between industry and academia for long term collaboration. Better stem cell differentiation protocols in mammalians will potentially allow for validation and development of iPS cells as highly desirable alternatives for live animals for drug and toxicity testing, disease modeling and research on regenerative medicine.
AniStem has allowed the consortium members to develop beyond the current state-of-the-art novel methods for producing iPS cells of rabbit and pig origin. This potentially will create a platform for future research on drugs to delay or even stop disease onset in humans and animals.