THE ESTABLISHMENT AND CHARACTERIZATION OF GENETICALLY ENGINEERED IPS CELL LINES DERIVED FROM HUMAN SKIN FIBROBLASTS
KUUSELA, JUKKA (2011)
2011
Biokemia - Biochemistry
Biolääketieteellisen teknologian yksikkö - Institute of Biomedical Technology
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Hyväksymispäivämäärä
2011-03-15
Julkaisun pysyvä osoite on
https://urn.fi/urn:nbn:fi:uta-1-21237
https://urn.fi/urn:nbn:fi:uta-1-21237
Tiivistelmä
The human pluripotent stem cells are able to differentiate into all cell types of the three primary germ layers. Genetically modified human pluripotent stem cells represent a powerful tool to study stem cell characteristics such as differentiation. However, the genetic modification of human pluripotent stem cells has been technically challenging unlike with differentiated cells. With the emergence of iPS cell technology, differentiated cells can now be reprogrammed into stem cell-like state. The aim of this study was to establish genetically modified human iPS cell lines by first genetically modifying human skin fibroblasts before reprogramming to iPS cells. Genetically modified iPS cells and their derived cardiomyocytes would be visualized by fluorescent reporters.
SIN-lentiviral vectors were used to genetically modify human skin fibroblasts. Retroviral pMX-vectors were used for human iPS cell reprogramming. Cell lines were characterized by immunocytochemistry and reverse-transcriptase PCR. Human iPS cells were differentiated into cardiomyocytes by co-culturing with END-2 cells. Human iPS cell pluripotency was assessed in vitro by embryoid body formation and analyzing germ layer markers by reverse-transcriptase PCR.
Several fluorescent human iPS cell lines were successfully established. However, the fluorescence was extinguished in one cell line. Human iPS cell lines showed positive immunocytochemical staining for stem cell specific proteins. The endogenous pluripotency genes were expressed. The retroviral exogenous transgenes were not silenced. Thus, the human iPS cell lines were partially reprogrammed. Despite partial reprogramming, the differentiation to cardiomyocytes was successful, but no fluorescent cardiomyocytes were obtained. The cardiomyocytes expressed cardiac specific proteins. The embryoid bodies expressed markers from all the three germ layers indicating pluripotency of the iPS cells.
Fluorescent human iPS cell lines, albeit partially reprogrammed were successfully established. However, as in hESCs, the transgene silencing phenomenon was also observed in iPS cells probably due to SIN-lentiviral based genetic modification. The aim to generate fluorescent cardiomyocytes was not successful. Despite of this, valuable information was acquired for future studies.
Asiasanat:human induced pluripotent stem cells, genetically modified stem cells, iPS cell reprogramming
SIN-lentiviral vectors were used to genetically modify human skin fibroblasts. Retroviral pMX-vectors were used for human iPS cell reprogramming. Cell lines were characterized by immunocytochemistry and reverse-transcriptase PCR. Human iPS cells were differentiated into cardiomyocytes by co-culturing with END-2 cells. Human iPS cell pluripotency was assessed in vitro by embryoid body formation and analyzing germ layer markers by reverse-transcriptase PCR.
Several fluorescent human iPS cell lines were successfully established. However, the fluorescence was extinguished in one cell line. Human iPS cell lines showed positive immunocytochemical staining for stem cell specific proteins. The endogenous pluripotency genes were expressed. The retroviral exogenous transgenes were not silenced. Thus, the human iPS cell lines were partially reprogrammed. Despite partial reprogramming, the differentiation to cardiomyocytes was successful, but no fluorescent cardiomyocytes were obtained. The cardiomyocytes expressed cardiac specific proteins. The embryoid bodies expressed markers from all the three germ layers indicating pluripotency of the iPS cells.
Fluorescent human iPS cell lines, albeit partially reprogrammed were successfully established. However, as in hESCs, the transgene silencing phenomenon was also observed in iPS cells probably due to SIN-lentiviral based genetic modification. The aim to generate fluorescent cardiomyocytes was not successful. Despite of this, valuable information was acquired for future studies.
Asiasanat:human induced pluripotent stem cells, genetically modified stem cells, iPS cell reprogramming