Substitution of the mouse tumorigenicity assay with a complete in vitro microRNA based assay for testing malignancy of human induced pluripotent stem cells

Ronde 2016 Module Maatschappelijke Partners: Het herstellend vermogen van menselijke organen is zeer beperkt zo niet afwezig. Met als gevolg het optreden van ziektes als diabetes en hartfalen wanneer maar voldoende orgaan specifieke cellen door schade uitvallen. Orgaantransplantatie kan dan tot genezing leiden, maar volwassen en foetaal donor weefsel is slechts beperkt beschikbaar. Humane geïnduceerde pluripotente stamcellen (hiPSCs) kunnen net als embryonale stamcellen in kweek vermeerderd worden en daarna worden aangezet zich te specialiseren tot een van ongeveer 220 cel types van het menselijk lichaam. hiPSCs kunnen van iedere persoon geïsoleerd en gekweekt worden en na specialisatie terug getransplanteerd worden. Er treedt dan geen afstotingsreactie op. Daarom zijn hiPSCs de ideale kandidaat cellen voor regeneratieve geneeskunde. Echter, ze kunnen zich ook ontwikkelen tot kwaadaardige tumoren. Daarom worden hiPSCs eerst getest in muizen om te zien of ze tumoren ontwikkelen. In dit project wordt een proefdiervervangende test ontwikkeld die in potentie ook nog informatiever is.

Samenvatting van de aanvraag
In 2006/2007 it was found that human induced pluripotent stem cells (hiPSCs) can be generated from somatic cells (e.g. skin fibroblasts, blood cells, cells isolated from urine) by overexpression of four transcription factors (OCT3/4, SOX2, KLF4, c-MYC). Similar to human embryonic stem cells (hESCs), hiPSCs can be maintained in culture indefinitely (self-renewal) and differentiate into virtually all cell types of the human body (pluripotency), e.g. neurons, pancreatic cells, heart muscle cells or retinal pigmented epithelial cells. Since they capture the genotype of the donor, hiPSCs are already used successfully for studying mechanisms of disease and for drug testing. In the near future hiPSC-derivatives hold great promise for being applied also in regenerative medicine, e.g. for treatment of degenerative diseases such as Parkinson’s, diabetes or cardiac infarction. In fact several pre-clinical and clinical trials are already ongoing or being planned for the near future. The fact that hiPSCs can be generated from any human individual makes the concept of personalized medicine a realistic goal. However the presence of residual, potentially malignant cells among the cell population being transplanted represents a serious safety issue and requires rigorous testing and identification of unsuitable hiPSC lines beforehand. Currently, the potential tumorigenicity of hiPSCs is tested by injecting cells into mice where they either form benign teratomas or malignant teratocarcinomas. However the Teratoma tumorigenicity assay is animal-dependent, costly, time-consuming and only qualitative and has never been standardized. Since the number of hiPSC lines is steadily rising and (pre)clinical studies are already on their way there is an urgent need for a new quantitative gold standard assay which is animal-independent. In our proposal we aim at developing and validating an in vitro assay based on microRNAs which is able to predict the tumorigenicity of hiPSCs and can therefore replace the Teratoma assay. Our preliminary data shows that microRNAs serving for diagnosis of germ cell tumors in humans are upregulated in the serum of mice with malignant hiPSC-derived tumors. By contrast microRNA levels were low in the serum of mice with benign hiPSC-derived tumors. Interestingly we also found increased levels of the same microRNA in the cell culture media of malignant hiPSCs in culture. This exciting result lays the foundation for developing a simple, fast and quantitative screening assay for microRNAs marking malignant hiPSCs based on quantitative PCR. Such an assay would significantly reduce the number of animals required for quality testing of hiPSCs and help to distinguish hiPSCs which are suitable for clinical applications from those which would be potentially dangerous. In the future the assay would allow the development of possible malignancies to be monitored in the blood of patients after transplantation with hiPSC-derived cell types.
Onderdeel van programma:
Gerelateerde subsidieronde:
Projectleider en penvoerder:
Dr. D.C.F. Salvatori PhD
Verantwoordelijke organisatie:
Leiden University Medical Center