The derivation of induced pluripotent stem cell (iPSC) lines from a wide range of mammalian species provides a valuable resource for comparative developmental studies. Routine reprogramming of human patient cells is being used to capture the genetic diversity of individuals to generate new cell models that can recapitulate developmental and disease processes in a dish. This strategy can be extended across genera to generate putative iPSCs from a wide range of mammalian species, using a common set of core reagents to select for pluripotent cells derived under conditions similar to those used for human iPSCs. The conservation of identity of key transcription factors is sufficient such that transduction with vectors expressing the human Oct4, Sox2, Klf4 and c-Myc cDNAs can reprogram somatic cells from a wide range of mammals without the need to clone species specific genes. A non-integrating Sendai RNA viral vector system was used to effectively reprogram fibroblasts from a range of felid species (Domestic and Asian Leopard Cat, Cheetah) and Non-Human Primate species (Rhesus Macaque and Gorilla) into stable, putative iPSC lines. While species-specific differences may exist in growth factor dependence during embryonic development in vivo, by using existing media systems that support conserved signalling pathways, putative iPSC lines were selected for that survived in vitro in human iPSC culture conditions. Of practical utility, these putative iPSC lines grow rapidly and can be cultured well beyond the point at which the parental fibroblast lines undergo senescence. The majority of putative iPSC lines derived by this non-integrating method maintain a normal diploid karyotype determined by Giemsa banding analysis. Standard in vitro and in vivo assays are underway to test the differentiation capabilities of these cell lines. Non-integrating, Sendai viral vector-based reprogramming provides a standardized and reproducible approach to generating viable animal cell models free of DNA-artefacts that complement human genome resource and cell banking efforts.
A conserved approach to non-integrated reprogramming of mammalian cells
Ileana Zucchi;
2017
Abstract
The derivation of induced pluripotent stem cell (iPSC) lines from a wide range of mammalian species provides a valuable resource for comparative developmental studies. Routine reprogramming of human patient cells is being used to capture the genetic diversity of individuals to generate new cell models that can recapitulate developmental and disease processes in a dish. This strategy can be extended across genera to generate putative iPSCs from a wide range of mammalian species, using a common set of core reagents to select for pluripotent cells derived under conditions similar to those used for human iPSCs. The conservation of identity of key transcription factors is sufficient such that transduction with vectors expressing the human Oct4, Sox2, Klf4 and c-Myc cDNAs can reprogram somatic cells from a wide range of mammals without the need to clone species specific genes. A non-integrating Sendai RNA viral vector system was used to effectively reprogram fibroblasts from a range of felid species (Domestic and Asian Leopard Cat, Cheetah) and Non-Human Primate species (Rhesus Macaque and Gorilla) into stable, putative iPSC lines. While species-specific differences may exist in growth factor dependence during embryonic development in vivo, by using existing media systems that support conserved signalling pathways, putative iPSC lines were selected for that survived in vitro in human iPSC culture conditions. Of practical utility, these putative iPSC lines grow rapidly and can be cultured well beyond the point at which the parental fibroblast lines undergo senescence. The majority of putative iPSC lines derived by this non-integrating method maintain a normal diploid karyotype determined by Giemsa banding analysis. Standard in vitro and in vivo assays are underway to test the differentiation capabilities of these cell lines. Non-integrating, Sendai viral vector-based reprogramming provides a standardized and reproducible approach to generating viable animal cell models free of DNA-artefacts that complement human genome resource and cell banking efforts.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
