A stem cell-based approach to the treatment of devil facial tumour disease (DFTD) in the Tasmanian devil (Sarcophilus harrisii)

Abstract

The study of marsupial biology has both intrinsic and comparative significance. Unique to the island of Tasmania, the Tasmanian devil (Sarcophilus harrisii) is the worldrs largest extant carnivorous marsupial and is on the verge of extinction in the wild due to a transmissible cancer termed devil facial tumour disease (DFTD). Stem cells, particularly induced pluripotent stem cells (iPSCs), offer unprecedented potential for future cellular therapies. The aim of our research is to explore the possibility of using stem cell technology to eradicate tumour cells from Tasmanian devils suffering from DFTD.nIn the first study of this thesis, we demonstrate the generation of Tasmanian devil induced pluripotent stem cells (DeviPSCs), the first described marsupial pluripotent stem cells, from devil dermal fibroblasts by lentiviral delivery of human transcription factors. Induced pluripotent stem cells are derived from adult somatic cells that have been re-programmed to a pluripotent state, akin to that observed in the early pre-implantation embryo, thus providing a powerful research tool. DeviPSCs display characteristic pluripotent stem cell colony morphology, with individual cells having a high nuclear to cytoplasmic ratio and alkaline phosphatase (AP) activity. Moreover, DeviPSCs are leukemia inhibitory factor (LIF) dependent and have reactivated endogenous OCT4 (POU5F1), POU2 (POU5F3), SOX2, NANOG and DAX1 genes, retained a normal karyotype, and concurrently silenced exogenous human transgenes. Notably, co-expression of both OCT4 and POU2, a paralogue of OCT4, suggests that they are the equivalent to cells of the epiblast, the marsupial equivalent of the eutherian inner cell mass (ICM). DeviPSCs readily formed embryoid bodies (EBs) and in vitro teratomas containing derivatives of all three embryonic germ layers. Thus, DeviPSCs demonstrate the highly conserved role of the reprogramming factors across the therian infraclasses and provide a powerful tool for future research into marsupial pluripotency and comparative developmental biology.nAs described in the second chapter of this thesis, we have generated Tasmanian devil mesenchymal stem cells (iMSCs) from DeviPSCs, thus providing an unlimited source of devil MSCs and circumventing the need to harvest tissues from live animals. MSCs are of considerable research interest in translational medicine due to their immunomodulatory, anti-inflammatory and regenerative properties coupled with a minimal teratogenicity. DeviPSCs were differentiated into MSCs (iMSCs) through both embryoid body formation assays (EB-iMSCs) and through inhibition of the transforming growth factor beta (TGF-b)/activin signalling pathway (SB-iMSCs). Both EB-iMSCs and SB-iMSCs are highly proliferative and express the common MSC characterization surface markers CD73, CD90 and CD105, in addition to the pluripotency transcription factors OCT4, SOX2 and NANOG. Expression of the marsupial pluripotency factor POU2 is significantly reduced in association with the transition from pluripotency to multipotency. Devil iMSCs readily differentiate along the adipogenic, osteogenic and chondrogenic pathways in vitro, confirming their intrinsic trilineage differentiation potential. Importantly, in vitro teratoma assays confirmed their multipotency, rather than pluripotency, since the iMSCs only formed derivatives of the mesodermal germ layer. Moreover, iMSCs show a tropism towards medium conditioned by devil facial tumour cells, and express a wide range of immunomodulatory and anti-inflammatory factors. Therefore, devil iMSCs will be a valuable tool for further studies on marsupial biology and may facilitate the development of an MSC-based treatment strategy against DFTD.In the third chapter of this study, we explored the possible use of devil iMSCs as an effective MSC-based treatment strategy against DFTD. Interestingly, conditioned medium derived from devil iMSCs significantly inhibited the proliferation of devil facial tumour cells from the two types of DFTD, termed DFT1 and DFT2. Recent research on human MSCs has shown them to be potent regulators of inflammation and the immune response, and to have both pro- and anti-tumour activity. Therefore, we assessed the effect of devil iMSCs on devil facial tumour cells in vitro. Upon co-culture with DFT cells, devil iMSCs had significantly elevated levels of key immunomodulatory and anti-inflammatory factors. Further, SB-iMSCs express devil IFNg, prompting studies to explore the possibility of using SB-iMSCs to restore the expression of MHC class I in DFT cells upon co-culture. Thus, devil iMSCs, warrant further research as a potential treatment against DFTD.n