Rapid generation of marsupial induced pluripotent stem cells suitable for use in next-generation conservation
- 1University of Melbourne
Abstract
Marsupials account for a disproportionate share of recent and impending mammalian extinctions, that has prompted investment in the development of "next generation" conservation biotechnologies. These include the generation of genetically selected/modified individuals for improved population fitness. Given the inherent limitations of working with threatened species, a flexible vehicle to develop these technologies is through the generation and editing of induced pluripotent stem cells (iPSCs). However, it is deemed necessary to derive transgene-free iPSCs for conservation target species, which can be highly time consuming and expensive. This study describes the generation of iPSCs from the marsupial fat-tailed dunnart (diPSCs) that serves as a model species for next generation conservation initiatives. These diPSCs exhibit features of totipotency when cells are maintained with exogenous reprogramming factors for long periods, until differentiation potential is required. Neonatal dunnart fibroblasts were reprogrammed using doxycycline-inducible Oct4, Sox2, Klf4, Lin28 and Myc, and the resulting iPSC lines were characterised at the transcriptomic, epigenetic and functional level (trilineage differentiation, embryoid body formation and mouse blastocyst injection assays). Dunnart iPSCs exhibited robust transcriptional and epigenetic activation of core pluripotency factors (POU5F1, POU5F3, SALL4) as well as factors associated with the naive and primed states of eutherian pluripotency (TBX3, KLF2, TFCP2L1, SOX11, DNMT3A). Dunnart iPSCs readily differentiated in trilineage and embryoid body formation assays, where they expressed markers of all three germ lineages. When injected into 8-cell mouse blastocysts, dunnart iPSCs integrated into both the inner cell mass and trophoblast, reflecting a capacity to contribute to embryonic and extra embryonic lineages during early development. Further, we show that diPSCs can maintain their potential in the presence of exogenous pluripotency factors, until required for differentiation. This robust iPSC generation and maintenance approach is a valuable avenue for the rapid application of next-generation conservation technologies in threatened species.
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BibTeX
@misc{tarulli2026rapid,
title = {{Rapid generation of marsupial induced pluripotent stem cells suitable for use in next-generation conservation}},
author = {G. A. Tarulli and D. K. Hansen and P. Adithya and P. R. Tatt and L. Zheng and N. D. Kolaitis and K. Filiminow and E. Scase and E. Cottingham and P. Jaichander and S. J. Hoyt and G. A. Hartley and N. Tillquist and R. J. O'Neill and N. A. Youngson and B. R. Carone and S. Ord and A. J. Pask and S. R. Frankenberg},
howpublished = {bioRxiv},
year = {2026},
month = nov,
doi = {10.64898/2026.11.07.765090},
url = {https://www.biorxiv.org/content/10.64898/2026.11.07.765090v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Rapid generation of marsupial induced pluripotent stem cells suitable for use in next-generation conservation
AU - G. A. Tarulli
AU - D. K. Hansen
AU - P. Adithya
AU - P. R. Tatt
AU - L. Zheng
AU - N. D. Kolaitis
AU - K. Filiminow
AU - E. Scase
AU - E. Cottingham
AU - P. Jaichander
AU - S. J. Hoyt
AU - G. A. Hartley
AU - N. Tillquist
AU - R. J. O'Neill
AU - N. A. Youngson
AU - B. R. Carone
AU - S. Ord
AU - A. J. Pask
AU - S. R. Frankenberg
PY - 2026
DA - 2026/11/07
PB - bioRxiv
DO - 10.64898/2026.11.07.765090
UR - https://www.biorxiv.org/content/10.64898/2026.11.07.765090v1
AB - Marsupials account for a disproportionate share of recent and impending mammalian extinctions, that has prompted investment in the development of "next generation" conservation biotechnologies. These include the generation of genetically selected/modified individuals for improved population fitness. Given the inherent limitations of working with threatened species, a flexible vehicle to develop these technologies is through the generation and editing of induced pluripotent stem cells (iPSCs). However, it is deemed necessary to derive transgene-free iPSCs for conservation target species, which can be highly time consuming and expensive. This study describes the generation of iPSCs from the marsupial fat-tailed dunnart (diPSCs) that serves as a model species for next generation conservation initiatives. These diPSCs exhibit features of totipotency when cells are maintained with exogenous reprogramming factors for long periods, until differentiation potential is required. Neonatal dunnart fibroblasts were reprogrammed using doxycycline-inducible Oct4, Sox2, Klf4, Lin28 and Myc, and the resulting iPSC lines were characterised at the transcriptomic, epigenetic and functional level (trilineage differentiation, embryoid body formation and mouse blastocyst injection assays). Dunnart iPSCs exhibited robust transcriptional and epigenetic activation of core pluripotency factors (POU5F1, POU5F3, SALL4) as well as factors associated with the naive and primed states of eutherian pluripotency (TBX3, KLF2, TFCP2L1, SOX11, DNMT3A). Dunnart iPSCs readily differentiated in trilineage and embryoid body formation assays, where they expressed markers of all three germ lineages. When injected into 8-cell mouse blastocysts, dunnart iPSCs integrated into both the inner cell mass and trophoblast, reflecting a capacity to contribute to embryonic and extra embryonic lineages during early development. Further, we show that diPSCs can maintain their potential in the presence of exogenous pluripotency factors, until required for differentiation. This robust iPSC generation and maintenance approach is a valuable avenue for the rapid application of next-generation conservation technologies in threatened species.
ER -