Final Report Summary - INDUSTEM (Comparative stem cell research in mouse and humans)
Pluripotent stem cells can divide indefinitely and differentiate to all somatic cells of the body. They therefore present unprecedented opportunities for studying differentiation in early development and obtaining cells normally inaccessible to biopsy like those of heart and brain. These have potential applications in cell therapy and in studying how human organ cells respond to drugs. Sources of human pluripotent stem cells are often human embryos but more recently it has become possible to reprogram somatic cells to a pluripotent, embryonic-like state without embryo use. The discovery of direct cell reprogramming and induced pluripotent stem (iPS) cell technology opened up new avenues for the application of non-viral, transposon-based gene delivery systems. However, the problem has been that the genes required for reprogramming integrated permanently into the genome of the host cells, creating the risk of disrupting essential functions of endogenous genes or even creating an oncogenic risk. The main scientific issue that was addressed in the Industem project was testing a novel reprogramming method based on the sleeping beauty (SB) transposon, a highly advanced tool for versatile genetic manipulations in mammalian cells by DNA elements that can 'jump' in and out of the genome by expression of transposases. SB was used to introduce the four principle reprogramming genes into somatic cells. The two academic partners (Leiden University Medical Centre and Max Delbruck Center for Molecular Medicine), developed the technology and used it to reprogram human cells, while the industrial partner Biotalentum used the technology to reprogram mouse somatic cells from three different genetic backgrounds and later had personnel trained in the use of human induced pluripotent stem cells. This enabled them to participate in producing and characterising new lines based on the transposon technology.
The iPS cell reprogramming of mouse embryonic fibroblasts and human foreskin and foetal fibroblasts by transposition of OSKM (Oct4, Sox2, Klf4 and c-Myc) and OSKML (OSKM + Lin28) expression cassettes mobilized by the SB100X hyperactive transposase.
The efficiency of iPS cell derivation with SB transposon system was in the range of that obtained with retroviral vectors. Co-expression of the miRNA302/367 cluster together with OSKM significantly improved reprogramming efficiency and accelerated the temporal kinetics of reprogramming. The iPS cells displayed a stable karyotype and hallmarks of pluripotency including expression of stem cell markers and the ability to differentiate into embryoid bodies in vitro. We demonstrate Cre recombinase-mediated exchange allowing simultaneous removal of the reprogramming cassette and targeted knock-in of an expression cassette of interest into the transposon-tagged locus in mouse iPS cells. This strategy would allow correction of a genetic defect by site-specific insertion of a therapeutic gene construct into 'safe harbor' sites in the genomes of autologous, patient-derived iPS cells.
The work is resulted in a series of (partially joint) publications describing the generation of mouse induced pluripotent stem cells and their properties, new technology for the transposon constructs and the production of human induced pluripotent stem cells.
A major interest of one academic partner (LUMC) and Biotalentum is the use of these cells in applications related to the heart. As part of an exchange visit of an early researcher from Biotalentum, new human pluripotent stem cell lines from patients with genetic cardiac disease of the contractile apparatus were generated and a start made in examining their phenotype. The early researcher returned to Hungary after a long research visit to the host and is presently involved in establishing the infrastructure to generate new human stem cell lines from patients with other relevant diseases. Funding from Industem also led to the successful recruitment of an overseas researcher. With Industem support, this researcher was involved in creating, characterising and publishing another cardiac disease model, this time of the electrical system. This researcher has remained with the host institute following his recruitment and has gained valuable stem cell and management experience.
Essential for all of this research are efficient differentiation protocols for the production of cardiomyocytes. These protocols existed at the outset of the project but have been refined and improved as part of the Industem efforts. Most importantly, they are now based on defined culture reagents which are animal product free, a prerequisite for future clinical applications but also attractive for the pharmaceutical industry in developing platforms for drug discovery and safety pharmacology.
The iPS cell reprogramming of mouse embryonic fibroblasts and human foreskin and foetal fibroblasts by transposition of OSKM (Oct4, Sox2, Klf4 and c-Myc) and OSKML (OSKM + Lin28) expression cassettes mobilized by the SB100X hyperactive transposase.
The efficiency of iPS cell derivation with SB transposon system was in the range of that obtained with retroviral vectors. Co-expression of the miRNA302/367 cluster together with OSKM significantly improved reprogramming efficiency and accelerated the temporal kinetics of reprogramming. The iPS cells displayed a stable karyotype and hallmarks of pluripotency including expression of stem cell markers and the ability to differentiate into embryoid bodies in vitro. We demonstrate Cre recombinase-mediated exchange allowing simultaneous removal of the reprogramming cassette and targeted knock-in of an expression cassette of interest into the transposon-tagged locus in mouse iPS cells. This strategy would allow correction of a genetic defect by site-specific insertion of a therapeutic gene construct into 'safe harbor' sites in the genomes of autologous, patient-derived iPS cells.
The work is resulted in a series of (partially joint) publications describing the generation of mouse induced pluripotent stem cells and their properties, new technology for the transposon constructs and the production of human induced pluripotent stem cells.
A major interest of one academic partner (LUMC) and Biotalentum is the use of these cells in applications related to the heart. As part of an exchange visit of an early researcher from Biotalentum, new human pluripotent stem cell lines from patients with genetic cardiac disease of the contractile apparatus were generated and a start made in examining their phenotype. The early researcher returned to Hungary after a long research visit to the host and is presently involved in establishing the infrastructure to generate new human stem cell lines from patients with other relevant diseases. Funding from Industem also led to the successful recruitment of an overseas researcher. With Industem support, this researcher was involved in creating, characterising and publishing another cardiac disease model, this time of the electrical system. This researcher has remained with the host institute following his recruitment and has gained valuable stem cell and management experience.
Essential for all of this research are efficient differentiation protocols for the production of cardiomyocytes. These protocols existed at the outset of the project but have been refined and improved as part of the Industem efforts. Most importantly, they are now based on defined culture reagents which are animal product free, a prerequisite for future clinical applications but also attractive for the pharmaceutical industry in developing platforms for drug discovery and safety pharmacology.